A protein kinase activity was defined in cytosols and membranes of rat uterus by using two types of enzyme inhibitors. The heat stable protein kinase inhibitor inhibited the cAMP-stimulated protein kinase but not the basal activity measured in the absence of the cyclic nucleotide. However, this basal activity was inhibited by quercetin in a dose-dependent manner, whereas the cAMP-stimulated protein kinase was not inhibited by this bioflavonoid. Thus, quercetin can be used as a tool to define a specific fraction of cAMP-independent protein kinase activity in rat uterine cytosols and membranes. The mature rat uterus is characterized by short (4 days) cyclic changes in tissue growth. Cell proliferation expressed as total uterine DNA content is observed on proestrus, reaches its peak at estrus, and declines sharply during metestrus and diestrus. The quercetin-inhibited protein kinase activity in the membranes of this tissue also changes cyclically and precedes by one phase the cyclic change in tissue proliferation. The clearest effect was seen when the protein substrates for this protein kinase activity were endogenous membrane proteins partially solubilized by Triton X-100 treatment. In contrast no change was observed in cytosolic, quercetin-inhibited protein kinase activity. The cAMP-stimulated protein kinase activity decreased slightly on diestrus. These results support our working hypothesis that tissue proliferation in the uterus and other tissues such as rat mammary gland and mammary tumors, correlates with cAMP-independent, quercetin-inhibited, protein kinase activity in these tissues.