A recombinant strain, isolated following the transduction of an Escherichia coli recipient carrying the Salmonella typhimurium (SB) specificity genes with DNA from a donor having the Salmonella potsdam (SP) specificity, was shown [Bullas, L.R., Colson, C. & Van Pel, A. (1976) J. Gen. Microbiol. 95, 166-172] to have neither SB nor SP specificity but to encode a novel restriction specificity, SQ. The heteroduplex analysis of the hsdS (specificity) genes of the SB and SP restriction and modification systems described here identifies a conserved sequence of around 100 base pairs flanked by two nonhomologous regions each of approximately 500 base pairs. This organization parallels that previously deduced from the DNA sequences of the hsdS genes of the related E. coli K-12, B, and D restriction systems. The present heteroduplex analyses further show that the hsdS gene conferring the SQ specificity derives one nonhomologous region from the SB gene and the other from the SP gene, as predicted from genetic exchange within the conserved sequence. This finding supports the idea that two domains of an hsdS polypeptide, which are different for each specificity, may correlate with two regions of the DNA sequence recognized. It has been shown that the recognition sequences for E. coli K-12 and B each consist of two short oligonucleotide sequences interrupted by a nonspecific sequence. A similar organization is suggested for the Salmonella specificity systems, providing the potential for evolutionary diversification of restriction specificities as a result of recombination within the conserved sequence of the hsdS gene.