Mouse mammary tumor virus (MMTV) expression and MMTV-induced tumorigenesis were studied in a congenic line of the BALB/cHeA strain, termed BALB/c-Mtv-3+, that carries the Mtv-3 proviral gene. BALB/c-Mtv-3+ mice were free of milk-transmitted MMTV and did not spontaneously develop mammary tumors. A specific Mtv-3 expression was observed in the mammary gland and spleen, but not in other lymphoid tissues, such as thymus and bone marrow. This expression was hormone dependent, as shown by the increase of MMTV mRNA during pregnancy. At the protein level, large amounts of p28, but only traces of gp52, the main MMTV core and envelope antigens, respectively, were observed, in agreement with the already described "partial" expression of the Mtv-3 gene products. The presence of the 24S (3.8 kilobases) mRNA encoding the MMTV env antigens in the spleen and the low gp52 reactivity in lactating mammary glands showed that this noncoordinate expression was probably due to a defect in translation or posttranslational processing of env proteins. The susceptibility of BALB/c-Mtv-3+ to experimental MMTV infection was studied. The presence of Mtv-3 conferred to BALB/c mice resistance to MMTV infection, as shown by measuring viral antigens released in the milk of infected mice and by recording the incidence of early mammary tumors. The presence of a nontumorigenic endogenous MMTV gene was therefore protective against exogenous MMTV infection.