The increasing proportion of Streptococcus pneumoniae isolates with reduced susceptibility to penicillin has created an urgent need for therapeutic alternatives to some beta-lactam agents. Clindamycin is an antimicrobial agent with excellent bioavailability after oral administration which has been considered for the therapy of community-acquired pneumococcal otitis media. Using the Etest methodology, we have studied the in vitro susceptibility of 59 erythromycin-resistant strains of S. pneumoniae to clindamycin, penicillin, trimethoprim-sulfamethoxazole, and rifampin. The study also addressed the impact of the susceptibility test medium [Mueller-Hinton (MH) vs IsoSensitest (Iso), both 5% blood supplement] on the results. A total of 20 isolates (37%) displayed constitutive clindamycin resistance on Iso blood agar, compared with only 11 (22%) on MH blood agar. The remaining nine strains found to be clindamycin susceptible on MH manifested resistance only with erythromycin induction. Resistance to penicillin, rifampin, and trimethoprim-sulfamethoxazole in erythromycin-resistant isolates was 83%, 2%, and 85%-89% (medium dependent), respectively. These results indicate that the choice of susceptibility test medium affects the expression (constitutive or inducible) of macrolide-lincosamide-streptogramin (MLS) resistance in S. pneumoniae. In addition, the common assumption that erythromycin resistance in S. pneumoniae implies clindamycin resistance may need to be reconsidered and routine susceptibility tests (including induction if MH medium is used) should be considered for MLS-class drugs.