The sequence of the gene encoding a novel cefotaxime-hydrolyzing beta-lactamase (CTX-M-4) was determined. It was located in a plasmid harbored by a Salmonella typhimurium strain. CTX-M-4 was similar to the plasmidic cefotaxime-hydrolyzing beta-lactamases CTX-M-2 and Toho-1 and related to the chromosomal beta-lactamase of Klebsiella oxytoca. A Ser-237-->Ala substitution, introduced by site-directed mutagenesis, caused minor alterations in the interaction of CTX-M-4 with beta-lactams, reducing slightly the relative hydrolytic activity against cefotaxime and the susceptibility to inhibition by clavulanate.