Immunolocalization of a newly described isoform of p70s6k, termed p85s6k, demonstrated a predominantly nuclear location in rat embryo fibroblasts (REF-52), a compartment in which growth factor-mediated phosphorylation of S6 has recently been reported. Microinjection of expression vectors encoding either p85s6k or a fusion protein containing only the putative nuclear localization motifs led to the exclusive accumulation of both products in the nucleus. Consistent with such a localization, microinjection of affinity-purified anti-p85s6k IgG into the nucleus, but not the cytoplasm, blocked serum-induced initiation of DNA synthesis. Co-injection into the nucleus of the anti-p85s6k IgG with activated p70s6k, which lacks the antigenic epitope, rescued the S phase block, arguing that the antibody exerts its effects through inhibiting p85s6k function. The results indicate a novel role for S6 phosphorylation in the nucleus distinct from that in the cytoplasm, a role essential for mitogenesis.