Derivatives of Streptococcus pneumoniae type 3 deficient in production of either pneumolysin or autolysin were constructed. This was achieved by transformation of type 3 pneumococci with DNA from derivatives of a rough strain (Rx1), in which the respective genes had been interrupted by insertion-duplication mutagenesis using internal fragments of the cloned genes in the vector pVA891. Southern blot analysis confirmed that the pneumolysin or autolysin genes in the respective transformants had been interrupted by insertion of the plasmid-derived sequences. Both the pneumolysin-negative and the autolysin-negative strains had significantly reduced (P less than 0.0001) virulence in mice, as judged by survival time after intraperitoneal challenge. The median survival time of mice challenged with type 3 pneumococci in which either pneumolysin or autolysin production had been reconstituted by back-transformation of the mutants with an intact copy of the respective cloned gene (with concomitant elimination of plasmid-derived sequences), was indistinguishable from that of mice challenged with the wild-type strain. These results establish the importance of both pneumolysin and autolysin to the virulence of type 3 pneumococci.