A total of 1561 pneumococcal isolates were collected in 1997–2001, mainly from patients with community-acquired respiratory tract infections, and susceptibilities were tested by reference broth microdilution against 29 antimicrobial agents. In general, 69.3% of strains were considered susceptible (MIC ≤ 0.06 mg/L) to penicillin. Resistance to penicillin (MIC ≥ 2 mg/L) and cefotaxime (MIC ≥ 4 mg/L) was found in 11.9% and 0.4% of isolates, respectively. The fluoroquinolones gatifloxacin (MIC90, 0.5 mg/L) and levofloxacin (MIC90, 1 mg/L) were active against > 99% of the isolates tested. Among the other non-β-lactam drugs tested, the rank order of susceptibility was chloramphenicol (95.6%) > clindamycin (94.5%) > azithromycin (88.5%) > clarithromycin (87.5%) >tetracycline (79.5%) > trimethoprim + sulphamethoxazole (60.5%). The penicillin-non-susceptible isolates presented higher rates of resistance to other antimicrobial agents. The rank order of penicillin resistance rates among the seven participating countries was Mexico (25.0%) > Uruguay (19.2%) > Chile (18.3%) > Colombia = Argentina (9.9%) > Brazil (3.9%) > Venezuela (2.8%). The regional rate of penicillin resistance did not vary significantly over the years studied (p 0.339). Screening for the ermB and mefA genes by multiplex rapid cycle PCR on 23 erythromycin-resistant isolates collected during the year 2001 showed that 43.5% and 56.5%, respectively, were positive for ermB and mefA. Overall, the results indicated that antimicrobial susceptibilities of Streptococcus pneumoniae vary significantly among Latin American countries. Regional and local surveillance programmes are necessary to guide empirical therapy of pneumococcal infection in Latin American countries.