We have recently shown that the estrogen-induced protein of rat uterus (IP) is indistinguishable from a constitutive protein in rat brain, and that the brain type gamma gamma isozyme of enolase (EC 4.2.1.11) is a component of IP. Here we report that the brain type BB isozyme of creatine kinase (EC 2.7.3.2) is the major component of IP. The two IP components, creatine kinase BB and enolase gamma gamma, were copurified from rat brain by ammonium sulfate fractionation and DEAE-cellulose chromatography. The enzymes were separated on reactive blue 2 agarose, which absorbs creatine kinase BB but not enolase gamma gamma, at 40 mMNaCl, pH 5.2. The major component of IP was identified as the BB isozyme of creatine kinase on the basis of its specific enzyme activity, chromatographic behavior, and specific immunoprecipitation by anti-creatine kinase BB antiserum. The identity of the major component of IP and creatine kinase BB was confirmed by double isotope ratio analysis, limited protease digestion patterns, and the rapid increase in the rate of synthesis of creatine kinase in rat uterus in response to estrogen. IP has been a favored marker for estrogen action in rat uterus because of its early response to the hormone both in vivo and in vitro. The identification of the major component of IP as the BB isozyme of creatine kinase is a step toward understanding the function of IP in the early responses of the uterus to estrogen and reveals a further advantage for IP as a marker for the study of gene expression, and as a possible enzymic marker for hormone responsive tumors.