Organisms producing extended-spectrum beta-lactamases (ESBLs) have been reported in many countries, but there is no information on the prevalence of ESBL-producing members of the family Enterobacteriaceae in Cameroon. A total of 259 Enterobacteriaceae strains were isolated between 1995 and 1998 from patients at the Yaounde Central Hospital in Cameroon. Enterobacterial isolates resistant to extended-spectrum cephalosporin and monobactam were screened for ESBL production by the double-disk (DD) synergy test. Thirty-one (12%) of these Enterobacteriaceae strains were shown to be positive by the DD synergy test, suggesting the presence of ESBLs. Resistance to oxyimino-cephalosporins and monobactams of 12 (38.7%) of the 31 strains-i.e., 6 Klebsiella pneumoniae, 4 Escherichia coli, 1 Citrobacter freundii, and 1 Enterobacter cloacae strain-was transferred to E. coli HK-225 by conjugation. Resistance to gentamicin, gentamicin plus trimethoprim-sulfamethoxazole, or trimethoprim-sulfamethoxazole was cotransferred into 6, 2, and 1 of these transconjugants, respectively. All 12 transconjugants were resistant to amoxicillin, piperacillin, all of the cephalosporins, and aztreonam but remained susceptible to cefoxitin and imipenem. Crude extracts of beta-lactamase-producing transconjugants were able to reduce the diameters of inhibition zones around disks containing penicillins, narrow- to expanded-spectrum cephalosporins or monobactams when tested against a fully susceptible E. coli strain but had no effect on such zones around cefoxitin, imipenem, and amoxicillin-clavulanate disks. The beta-lactamases produced by the 12 tranconjugants turned out to be SHV-12 by DNA sequencing. Therefore, the ESBL SHV-12 is described for the first time in Cameroon.