HHblits is a fast, iterative protein sequence search tool that uses profile hidden Markov models (HMMs) for improved sensitivity and accuracy. It is faster than PSI-BLAST and more sensitive, generating more accurate alignments. HHblits builds multiple sequence alignments (MSAs) by iteratively searching through sequence databases, which are crucial for predicting evolutionary conserved properties like protein structure and function. It uses a discretized profile-column alphabet to reduce the number of full HMM-HMM alignments, making it faster than PSI-BLAST but as sensitive as HHsearch. HHblits also includes a prefilter that reduces the number of database sequences by up to 2,500-fold, improving efficiency. It is used in many protein structure prediction servers, including HHpred, which is top-ranked in the Critical Assessment of Techniques for Protein Structure Prediction. HHblits is faster than PSI-BLAST and HMMER3, with HHblits being about twice as fast as PSI-BLAST and 6× faster than HMMER3. It also shows higher sensitivity and precision in detecting homologous proteins. HHblits improves the accuracy of secondary structure prediction by using MSAs generated from HHblits. It is also used to predict structures for Pfam families with no known templates. HHblits is open-source and can be used for various downstream analysis and prediction methods. It requires databases of MSAs and their HMMs instead of single sequences, and customized databases need to be built specifically for HHblits. HHblits is a robust, general-purpose tool that is faster, more sensitive, and produces higher quality alignments than PSI-BLAST.HHblits is a fast, iterative protein sequence search tool that uses profile hidden Markov models (HMMs) for improved sensitivity and accuracy. It is faster than PSI-BLAST and more sensitive, generating more accurate alignments. HHblits builds multiple sequence alignments (MSAs) by iteratively searching through sequence databases, which are crucial for predicting evolutionary conserved properties like protein structure and function. It uses a discretized profile-column alphabet to reduce the number of full HMM-HMM alignments, making it faster than PSI-BLAST but as sensitive as HHsearch. HHblits also includes a prefilter that reduces the number of database sequences by up to 2,500-fold, improving efficiency. It is used in many protein structure prediction servers, including HHpred, which is top-ranked in the Critical Assessment of Techniques for Protein Structure Prediction. HHblits is faster than PSI-BLAST and HMMER3, with HHblits being about twice as fast as PSI-BLAST and 6× faster than HMMER3. It also shows higher sensitivity and precision in detecting homologous proteins. HHblits improves the accuracy of secondary structure prediction by using MSAs generated from HHblits. It is also used to predict structures for Pfam families with no known templates. HHblits is open-source and can be used for various downstream analysis and prediction methods. It requires databases of MSAs and their HMMs instead of single sequences, and customized databases need to be built specifically for HHblits. HHblits is a robust, general-purpose tool that is faster, more sensitive, and produces higher quality alignments than PSI-BLAST.