After growth to exponential phase in Todd-Hewitt broth, clinical and laboratory isolates of Streptococcus pneumoniae serotypes 3, 4, and 14 readily degraded first the β and then the α chains of purified human C3 in the absence of serum or other complement proteins, as assessed by SDS-PAGE. With exponentially growing pneumococci, degradation of native C3 was detectable within 30 min; methylamine-treated C3 and preformed C3b were degraded with equal avidity. Pneumococcal C3-degrading activity was cell associated, abolished by heat killing, and independent of the presence of the polysaccharide capsule. After degradation, 44% of C3 molecules contained a disrupted thiolester bond. Pneumococci treated with 100 'b5g of mutanolysin released 94% of C3-degrading activity from the pneumococcal surface into the supernatant. These studies demonstrate that clinical and laboratory isolates of virulent pneumococci degrade and inactivate soluble C3.