Two main mechanisms of macrolide resistance have been described in erythromycin-resistant Streptococcus pneumoniae (ERSP): a ribosomal methylase, ErmAM, and a macrolide efflux pump, MefE. In this study, we examined the prevalence of these mechanisms in 114 clinical isolates of ERSP from a 30-center study conducted in the United States between November 1994 and April 1995. The isolates were screened by polymerase chain reaction for the presence of known macrolide resistance genes. Seventy (61%) ERSP contained the macrolide efflux gene (mefE), whereas 36 isolates (32%) contained the biosomal methylase gene (ermAM). Isolates that were ermAM-positive had constitutive macrolide resistance. The minimum inhibitory concentrations (for which 90% of isolates were susceptible) of clarithromycin for the efflux-positive strains were much lower than those for the ermAM-positive strains (4 microg/mL vs. >128 microg/mL, respectively). The efflux mechanism is the predominant form of macrolide resistance in the United States.