A nationwide multicenter study (including 31 laboratories) of the antimicrobial susceptibility of 1210 Streptococcus pneumoniae isolates from patients with community-acquired lower respiratory tract infections (LRTI) was carried out over 3 years (1999-2001) in Portugal. Testing of all isolates was undertaken in a central laboratory. Overall macrolide resistance was 13.1%. Decreased susceptibility to penicillin was 24.5% (15.5% low-level and 9.0% high-level resistance). Taken into consideration, the resistance rates reported in a previous surveillance study of 1989-1993, a six-fold increase of erythromycin resistance in the last decade was documented. Resistance to erythromycin, clarithromycin, and azithromycin was higher in pediatric patients than in adults. The overwhelming majority (82.3%) of macrolide-resistant isolates were multidrug resistant, although 44.9% were fully susceptible to penicillin. Most macrolide-resistant isolates (80.4%) showed the MLSB phenotype (76.6% MLSB-constitutive resistance, and 3.8% MLSB-inducible resistance) and were also resistant to clindamycin, tetracycline, and co-trimoxazole. The M phenotype was seen in 19.6% isolates and these had MIC90 values of 8 mg/L for erythromycin and clarithromycin, and of 12 mg/L for azithromycin. The clinical significance of macrolide resistance in the management of LRTI is discussed. Because of the specific situation concerning macrolide resistance described in S. pneumoniae, careful use of macrolide antibiotics in therapy and cautious monitoring of macrolide resistance should be continued in Portugal.