Group B streptococci are emerging as a cause of serious infection worldwide. The capsular polysaccharides are not only important virulence factors but also the target of vaccine development efforts. Serotypes III (24.6%), V (23.4%), Ia (17.8%), and II (16.3%) were the most prevalent among 252 Streptococcus agalactiae isolates collected during 1999-2002 in the largest hospital of Lisbon, Portugal. The substantial proportion of bacteremic patients (17 neonates and 21 adults) in this period illustrates the present importance of S. agalactiae as a cause of invasive disease. All isolates were fully susceptible to penicillin (MIC(50) = 0.064 microg/ml; MIC(90) = 0.094 microg/ml, range 0.008-0.094), cefotaxime, chloramphenicol, ofloxacin, and vancomycin. Resistance was found to tetracycline (75.4%), erythromycin (10.7%), and clindamycin (9.9%). Of the 27 erythromycin-resistant isolates, 70.4% had the cMLS(B), 22.2% the iMLS(B), and 7.4% the M phenotype. All isolates presenting the M phenotype carried the mef(A) gene, whereas the erm(B) gene was found in a large fraction of MLS(B) isolates (n = 17) and only a small proportion (n = 7) the erm(A) gene [erm(TR) variant]. All isolates carried a single macrolide-resistance determinant. Macrolide resistance was not attributable to a single clone as evidenced by distinct serotype and pulsed-field gel electrophoretic profiles. Careful surveillance of S. agalactiae invasive infections in Portugal is essential, and the treatment or intrapartum prophylaxis of patients who are allergic to penicillin should be guided by contemporary resistance patterns observed in the country.