Streptococcus pneumoniae isolated at Children's Hospital of Wisconsin during the winter of 1994 to 1995 and the winter of 1996 to 1997 were tested for susceptibility to penicillin, cephalosporins and other potentially therapeutically useful antimicrobial agents to determine the prevalence of penicillin and multi-drug resistant isolates. During those years, the prevalence of S. pneumomiae not susceptible to penicillin was 27% and 28%, respectively, with 14% and 18%, respectively, of the respiratory isolates being high-level penicillin resistant. Despite the stable numbers of penicillin resistant isolates, there was evidence of significant increase in the resistance of these isolates to other antimicrobial agents. Respiratory isolates not susceptible to cefotaxime increased (p = .01; Fisher exact test) from 3% in 1995 to 20% in 1997. There was also a significant increase in the isolates not susceptible to erythromycin (p = .09; Fisher exact test) and trimethoprim/sulfamethoxazole (p < .01; Fisher exact test). This increase in resistance to multiple antimicrobial agents has significant implications for antibiotic therapy of children with infections likely to be due to Streptococcus pneumoniae.