To assess the role of capsular serotypes in the virulence of Streptococcus pneumoniae, we have constructed isogenic derivatives differing only in the type of capsule expressed. Strains of types 2, 5, and 6B were converted to type 3 by transformation and selection for an erythromycin resistance marker linked to the type 3 capsule locus. Characterization studies revealed that these type 3 derivatives were indistinguishable from the type 2, type 5, and type 6B parental strains in terms of restriction enzyme fragment pattern and expression of pneumococcal surface protein A (PspA). Expression of the type 3 capsule did not alter the mouse virulence of the similarly virulent type 2 strain. However, alteration of capsule type had a profound effect on the virulence of the type 5 and type 6B derivatives. The highly virulent type 5 strain was essentially avirulent when expressing the type 3 capsule. Conversely, the 50% lethal dose of the relatively avirulent type 6B strain was reduced >100-fold when the type 3 capsule was expressed. Thus, the serotype of capsule expressed has a major effect on virulence, and this effect is dependent upon the genetic background of the recipient strain.