TopPred II is an improved software for predicting the topology of membrane proteins. It is based on the previous freeware TOP-PRED and is now a standalone application written in Symantec THINK Pascal, allowing operation on any Macintosh computer with a 6.0.2 or later system. The program is compact (approximately 90 kilobytes) and includes all default parameters, scales, and texts as resources, enabling easy access and modification by the user. Input sequences are limited to 2000 amino acids, and sequences can be handled individually or in groups of up to 20. The program allows users to modify parameters and scales through standard dialogs, which also enable resetting to default values. It can calculate hydrophobicity profiles, transmembrane segments, and topologies. The results can be printed or saved in files for import by other programs. The prediction of membrane protein structure begins with constructing a hydrophobicity profile to identify transmembrane segments. This is done using a trapezoid sliding window, which is more realistic than a simple rectangular window. The GES hydrophobicity scale is recommended. Transmembrane domains are classified as 'certain' or 'putative' based on cutoff parameters. For prokaryotic proteins, the number of positively charged residues at inter-transmembrane segments is counted. Segments longer than the 'Critical Length' parameter are not considered, but the first N-terminal loop is always taken into account. The best topology is predicted using the 'positive-inside' rule. For eukaryotic proteins, three criteria are used to determine topology. These include differences in positively charged residues between the two sides of the membrane, net charge differences between N-terminal and C-terminal residues, and compositional distance analysis of long loops. The program also allows reducing the unfavorable free energy of membrane insertion of charged residues using the 'Charge-pair Energy' parameter. TopPred II predicts the topologies of 95% of tested prokaryotic proteins and 83% of tested eukaryotic proteins. It can be obtained via anonymous FTP from the EMBL server or by contacting the authors. The program has been validated on proteins synthesized in the cytoplasm and inserted into the prokaryotic inner plasma membrane or the eukaryotic endoplasmic reticulum. It may also be useful for proteins synthesized in the mitochondrial matrix or chloroplast stroma.TopPred II is an improved software for predicting the topology of membrane proteins. It is based on the previous freeware TOP-PRED and is now a standalone application written in Symantec THINK Pascal, allowing operation on any Macintosh computer with a 6.0.2 or later system. The program is compact (approximately 90 kilobytes) and includes all default parameters, scales, and texts as resources, enabling easy access and modification by the user. Input sequences are limited to 2000 amino acids, and sequences can be handled individually or in groups of up to 20. The program allows users to modify parameters and scales through standard dialogs, which also enable resetting to default values. It can calculate hydrophobicity profiles, transmembrane segments, and topologies. The results can be printed or saved in files for import by other programs. The prediction of membrane protein structure begins with constructing a hydrophobicity profile to identify transmembrane segments. This is done using a trapezoid sliding window, which is more realistic than a simple rectangular window. The GES hydrophobicity scale is recommended. Transmembrane domains are classified as 'certain' or 'putative' based on cutoff parameters. For prokaryotic proteins, the number of positively charged residues at inter-transmembrane segments is counted. Segments longer than the 'Critical Length' parameter are not considered, but the first N-terminal loop is always taken into account. The best topology is predicted using the 'positive-inside' rule. For eukaryotic proteins, three criteria are used to determine topology. These include differences in positively charged residues between the two sides of the membrane, net charge differences between N-terminal and C-terminal residues, and compositional distance analysis of long loops. The program also allows reducing the unfavorable free energy of membrane insertion of charged residues using the 'Charge-pair Energy' parameter. TopPred II predicts the topologies of 95% of tested prokaryotic proteins and 83% of tested eukaryotic proteins. It can be obtained via anonymous FTP from the EMBL server or by contacting the authors. The program has been validated on proteins synthesized in the cytoplasm and inserted into the prokaryotic inner plasma membrane or the eukaryotic endoplasmic reticulum. It may also be useful for proteins synthesized in the mitochondrial matrix or chloroplast stroma.