Klebsiella oxytoca clinical isolate A was recovered from the urine of a 55-year-old man with prostatic and urinary tract infections. This isolate displayed a beta-lactam resistance phenotype consistent with overproduction of a chromosomally encoded class A beta-lactamase and had decreased susceptibilities to all beta-lactams except ceftazidime, cephamycins, and carbapenems. Four weeks after treatment with an antibiotic regimen that included ceftazidime, K. oxytoca isolate B, which had a high level of resistance to ceftazidime, was isolated from the urine of the same patient. Isoelectric focusing analysis of the culture extracts of these isolates gave a pI of 5.4 for both isolates. Cloning experiments with the PCR products of the bla(OXY) gene resulted in two Escherichia coli DH10B recombinant clones with resistance phenotypes mirroring those of the parental isolates. Sequencing analysis revealed that the bla(OXY-2-5) gene from K. oxytoca B had a single nucleotide substitution compared to the sequence of the bla(OXY-2) gene from K. oxytoca A, leading to a proline-to-serine substitution at position 167, according to the numbering of Ambler. Biochemical analysis of purified OXY-2-5 showed that it had the ability to hydrolyze ceftazidime. This is the first report of in vivo selection of a K. oxytoca isolate that produced a chromosomally encoded beta-lactamase conferring resistance to ceftazidime.