Previous studies have shown that the envelope protein of the mouse mammary tumor virus (MMTV) rapidly upregulates prolactin (PRL) receptors by shifting them from internal pools to the cell surface and downregulates epidermal growth factor (EGF) receptors by inducing their internalization and degradation. This study shows that the effect on PRL receptors is mediated by the nitric oxide (NO)/cGMP pathway, since it can be mimicked by an NO donor or 8-bromo-cGMP and can be blocked by an NO synthase inhibitor. In contrast, the effect on EGF receptors is mediated by tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K), since it can be blocked by either a tyrosine kinase inhibitor or by a PI3K inhibitor. Both of these pathways can be activated by a calcium ionophore and inhibited by calcium chelation. Therefore, it appears that the mouse mammary tumor virus envelope protein, like other retroviral envelope proteins, initially elevates cytoplasmic calcium, which can then stimulate both the NO/cGMP and the tyrosine phosphorylation/PI3K pathways, leading to PRL receptor upregulation and EGF receptor downregulation, respectively.