The 011IO-1 ,B-lactamase gene was subcloned in a 1.16-kilobase TaqI fragment in the 2.4-kilobase chimeric plasmid pSK04. After directional subcloning into M13, the DNA sequence of this fragment was determined. The results showed an open reading frame of 858 base pairs (bp) encoding a protein of 286 amino acids. The structural gene showed 95, 87, and 60% DNA sequence identity with SHV-1, LEN-1, and TEM-1, respectively, and 93, 85, and 62% predicted amino acid sequence identity, respectively. The 87 bp upstream of the OHIO-i structural gene had 96% identity with the upstream flanking sequence of SHV-1, including the -35 and -10 consensus sequences and the putative ribosomal binding site. A 223-bp DNA probe derived from a Psfi-HaeHl fragment in the C-terminal sequence of OHIO-1 had predicted 96, 88, and 61% sequence identity with SHV-1, LEN-1, and TEM-1, respectively. This probe hybridized to SHV-1 and poorly to LEN-1, but not to TEM-1 or a variety of other plasmid-mediated I-lactamase genes, under stringent conditions. Screening of plasmid DNA derived from 40 ampicillin-resistant clinical isolates by Southern hybridization with the 223-bp probe uncovered no strains encoding OHIO-1. Isoelectric focusing of the same collection did identify two strains producing enzymes resembling SHV-1, however. We have also performed a kinetic comparison of OHIO-1, SHV-1, and TEM-1. OHIO-1 and SHV-1 were indistinguishable from each other but could be distinguished from TEM-i. These data clearly place OHIO-1 within the SHV-1 family of 13-lactamases.The OHIO-1 P-lactamase is a group 2b enzyme with a substrate and inhibitor profile resembling those of TEM-1 and SHV-1 (8, 28). Recent immunochemical data (34) and inhibitor profiling data (A. A. Medeiros, personal communication) suggest that OHIO-1 belongs within the SHV-1 family of P-lactamases. LEN-1 is a ,B-lactamase encoded on the chromosomes of some strains of Klebsiella pneumoniae with a pI of 7.0 and a nucleotide sequence resembling that of SHV-1 (2, 22). OHIO-1 has a pI similar to that of LEN-1 but is plasmid mediated. OHIO-1 appears to have been geographically restricted within the state of Ohio (17,28,29). To better understand the evolution of this restricted resistance mechanism, we have sequenced the structural gene and its flanking sequences. To arrive at a better understanding of structure and function among P-lactamases, we have searched for differences in substrate specificities among these enzymes and compared these with differences in their sequences. Finally, we have developed an intragenic DNA probe from the OHIO-1 gene sequence.