The nucleotide sequence of the rightward operator (OR) of phage λ was determined using simple and rapid DNA sequencing methods. The sequence of 74 base pairs of OR is presented, along with the base pair change caused by a mutation in OR. The methods described are applicable for sequencing DNA fragments with unique 5' ends up to 50 base pairs in length. Previous experiments showed that OR contains multiple sites recognized by the λ phage repressor. The authors identified three of these sites. Each of the two operators in the DNA of phage λ consists of multiple, nonidentical sites specifically recognized by the λ repressor. The sequence of the leftward operator (OL) was previously reported, and the base pair change caused by a mutation which reduces the affinity of OL for repressor was described. The sequence of OR was determined here, and the base pair change caused by a mutation in OR is also described. The methods used allow for the rapid determination of sequences of small (30–50 base pairs) double-stranded DNA molecules. Comparison of OR sequences with those in OL led to a model that identifies the sequences involved in repressor recognition. The study used restriction enzymes and DNA sequencing techniques to isolate and analyze fragments of OR. The sequences were determined by end labeling, nick-translation, and two-dimensional fractionation. The results showed that OR contains three repressor binding sites, each 17 base pairs long, separated by A+T-rich spacers. The sequence of OR was confirmed by analyzing endonuclease IV digestion products and comparing the results with previously determined sequences. The study also identified an operator mutant, VN, which showed a base pair change in OR. The results provide a detailed understanding of the structure and function of the OR region in phage λ.The nucleotide sequence of the rightward operator (OR) of phage λ was determined using simple and rapid DNA sequencing methods. The sequence of 74 base pairs of OR is presented, along with the base pair change caused by a mutation in OR. The methods described are applicable for sequencing DNA fragments with unique 5' ends up to 50 base pairs in length. Previous experiments showed that OR contains multiple sites recognized by the λ phage repressor. The authors identified three of these sites. Each of the two operators in the DNA of phage λ consists of multiple, nonidentical sites specifically recognized by the λ repressor. The sequence of the leftward operator (OL) was previously reported, and the base pair change caused by a mutation which reduces the affinity of OL for repressor was described. The sequence of OR was determined here, and the base pair change caused by a mutation in OR is also described. The methods used allow for the rapid determination of sequences of small (30–50 base pairs) double-stranded DNA molecules. Comparison of OR sequences with those in OL led to a model that identifies the sequences involved in repressor recognition. The study used restriction enzymes and DNA sequencing techniques to isolate and analyze fragments of OR. The sequences were determined by end labeling, nick-translation, and two-dimensional fractionation. The results showed that OR contains three repressor binding sites, each 17 base pairs long, separated by A+T-rich spacers. The sequence of OR was confirmed by analyzing endonuclease IV digestion products and comparing the results with previously determined sequences. The study also identified an operator mutant, VN, which showed a base pair change in OR. The results provide a detailed understanding of the structure and function of the OR region in phage λ.