OBJECTIVES: To enumerate and characterize extended-spectrum beta-lactamases (ESBLs) amongst ceftriaxone-resistant coliforms in Blantyre, Malawi, where third-generation cephalosporin use is currently highly restricted. METHODS: Over the period April 2004-March 2005 all ceftriaxone-resistant isolates from blood cultures were examined for the presence of ESBLs. Isoelectric focusing was performed on enzyme extracts. PCR and DNA sequencing of amplicons were used to identify the underlying genetic determinants responsible for the ESBL phenotypes. Transferability of the ESBL phenotypes was tested by conjugation to a susceptible Escherichia coli J53. RESULTS: Enterobacteriaceae were isolated from 1191 blood cultures, of which 19 (1.6%) were ceftriaxone resistant. Ten isolates (0.7% of all isolates) demonstrated an ESBL phenotype but only eight were characterized as three isolates were from the same patient. Genotypes SHV-11 (n = 1), SHV-12 (n = 3), SHV-27 (n = 1), TEM-63 (n = 2) and CTX-M-15 (n = 1) were detected. Plasmid transfer of the ESBL resistance phenotype was successful for all the isolates. CONCLUSIONS: In a clinical setting of minimal cephalosporin usage there is already a diversity of ESBL genotypes. Increased use of cephalosporins in this setting is likely to result in a rapid expansion of ESBLs and their prevalence will need to be carefully monitored.