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The antiphagocytic effect of M protein has been considered a critical element in virulence of the group A streptococcus. The hyaluronic acid capsule also appears to play an important role: studies of an acapsular mutant derived from the mucoid or highly encapsulated M protein type 18 group A streptococcal strain 282 indicated that loss of capsule expression was associated with decreased resistance to phagocytic killing and with reduced virulence in mice. To study directly the relative contributions to virulence of M protein and the hyaluronic acid capsule in strain 282, we inactivated the gene encoding the M protein (emm18) both in wild-type strain 282 and in its acapsular mutant, strain TX72. Inactivation of emm18 was accomplished by integrational plasmid mutagenesis, using the temperature-sensitive shuttle vector pJRS233 harboring a 5' DNA segment of emm18. As reported previously, wild-type strain 282 was resistant to phagocytic killing in vitro, both in whole human blood and in 10% serum. The capsule mutant TX72 was highly susceptible to phagocytic killing in 10% serum and moderately sensitive in whole blood. The M protein mutant 282KZ was highly susceptible to phagocytic killing in blood but only moderately sensitive in 10% serum. The double mutant TX74 was sensitive to killing in both conditions. In a mouse infection model, the 50% lethal dose was increased by 60- and 80-fold for the capsule and double mutants, respectively, compared with that of strain 282, but only by 6-fold for the M protein mutant. Integration of the strain 282 capsule genes into the chromosome of a nonmucoid M1 strain resulted in high-level capsule production and rendered the transformed strain resistant to phagocytic killing in 10% serum. These results provide further evidence that the hyaluronic acid capsule confers resistance to phagocytosis and enhances group A streptococcal virulence. The results suggest also that assessment of in vitro resistance to phagocytosis in 10% serum rather than in whole blood may be a more accurate reflection of virulence in vivo of group A streptococci.