From 1994 through 1999, the available isolates (4148 isolates) from active population-based surveillance of invasive pneumococcal disease in metropolitan Atlanta were serotyped and were tested for antimicrobial susceptibility. Macrolide-resistant isolates were studied for the presence of ermAM (a ribosomal methylase gene), mefE (a macrolide efflux gene), and tetM (the class M tetracycline resistance gene). Macrolide resistance increased from 16% of all invasive isolates in 1994 to 32% in 1999. Of the macrolide-resistant pneumococcal isolates studied, 99% contained genomic copies of mefE or ermAM. Isolates with ermAM were mainly serotypes 6B, 23F, 14, or 19F and contained tetM; mefE-associated isolates were predominantly serotypes 14, 6A, or 19F, and most did not contain tetM. The frequency of the ermAM-mediated phenotype in invasive Streptococcus pneumoniae remained stable over the 6-year surveillance. However, the mefE-mediated phenotype increased from 9% in 1994 to 26% of all isolates in 1999 and was noted in new serotypes. By 1999, 93% of the mefE-containing strains had minimum inhibitory concentrations >/=8 microgram/mL. Dissemination of the mefE determinant accounted for the rapid increase in the rate of macrolide resistance in our S. pneumoniae population.