We report here for the first time, that the SV40 small t-antigen inhibits mammary gland differentiation during mid-pregnancy and that about 10% of multiparous WAP-SVt transgenic animals develop breast tumors with latencies ranging from 10-17 months. Cyclin D1 is deregulated and over expressed in the small t-antigen positive mammary gland epithelial cells (ME-cells) and in the breast tumor cells. SV40 small t-antigen immortalized ME-cells (t-ME-cells) exhibit a strong intranuclear cyclin D1 staining, also in the absence of external growth factors and the cells continue to divide for several days without serum. In addition, the expression rate of cyclin E and p21(Waf1) but not of p53 is increased. Coimmunoprecipitation experiments revealed that p21(Waf1) is mainly associated with the cyclin D/CDK4 but not with the cyclin E/CDK2 complex. WAP-SVT transgenic animals exhibit an almost regular mammary gland development until late pregnancy but the majority of the ME-cells are eliminated by apoptosis during the early lactation period. Tumor formation is delayed and less efficient than in T/t-antigen positive animals. Sequestration of p53 and pRb by the N-terminal truncated T-antigen molecules (T1-antigen and T2-antigen) does not affect mammary gland differentiation and the transgenic animals (WAP-SVBst-Bam) do not develop breast tumors.