The cellfree supernatant from cultured splenocytes contains an immunogenic molecule capable of inducing anti-DNA antibodies in vitro and in vivo in young preautoimmune NZB/W and some normal mouse strains. The response to this supernatant material does not reflect polyclonal activation, and the molecule responsible for its immunogenic activity appears to be a relatively small (approximately 150 base pairs) DNA molecule, predominantly (75 to 80%) double stranded with tail-end single-strand regions. It does not appear to contain any unusual secondary structure. This immunogenic DNA molecule can be recovered from cultures of some normal as well as young B/W mice. It contains no observable protein moiety, and after phenol-chloroform extraction, the immunogenic DNA migrates on polyacrylamide and agarose gels as a relatively narrow homogeneous band. The anti-DNA antibody response detected in the serum of mice after injection of this DNA molecule appears to have its greatest affinity for the homologous DNA and for undenatured calf thymus DNA. Unique base sequences do not appear to be responsible for its immunogenic effects. Rather, this may depend on its relatively small size, since calf thymus DNA sheared to a similar size and also found incapable of digestion with restriction endonucleases had a similar immunogenic effect.