Previous in vitro studies had suggested that a product of the env gene of murine MCF (polytropic)-related sequences plays a role in regulating lymphocyte activation. To determine whether such an effect occurs in vivo, we have studied mice injected with phosphorothioate oligonucleotides antisense to such sequences. Injection of mice with antisense to the initiation region of the env gene resulted in (i) increased spleen cell numbers, primarily due to an increase in splenic B cells, (ii) increased class II MHC expression on B cells, (iii) increased RNA and DNA synthesis, and (iv) increased numbers of Ig producing cells. These results obtained with the antisense to MCF-related env did not occur with two scrambled phosphorothioate oligonucleotides or with antisense oligonucleotides to the initiation region of the env gene of xenotropic or ecotropic retroviral sequences. These data suggest that products of certain endogenous retroviral sequences regulate lymphocyte activation in vivo.