One of the earliest responses of the rat uterus to estrogen is increased synthesis of a specific cytosol protein (IP). This synthesis is detectable within 40 min and is dependent on an even earlier actinomycin D-sensitive function. IP has now been purified to homogeneity as determined by SDS polyacrylamide gel electrophoresis (SDS PAGE). The procedure was complicated by a tendency of the more homogeneous and concentrated material to aggregate. Purification consisted of sequential chromatography, in the presence of 0.1% triton X-100, via DEAE-cellulose (2x), hydroxylapatite and agarose-acrylamide. These were followed by preparative PAGE and finally SDS PAGE. Molecular weight determination by Ferguson plot analysis yielded an apparent molecular weight of 45 000. On final SDS PAGE, the material consisted of two major bands: the 45 000 molecular weight IP band and a band with an estimated molecular weight of 80 000. This second band displayed an elevated synthesis in E2-stimulated uteri similar to IP and appeared to consist of some form of aggregated IP. Carbohydrate determination on SDS gels using periodic acid-Schiff (PAS) stain was negative. Co-purification of labeled cytosol proteins from uteri of control and E2-stimulated rats revealed that IP is synthesized to some extent in the unstimulated animal as well as the stimulated.