The major objective of this study was to make a polyclonal antibody to a previously described group of progesterone (P)-dependent low molecular weight secretory proteins of the cat uterus. Proteins present in uterine flushings obtained from a P-treated cat were partially purified using Sephadex G-75, separated on two-dimensional polyacrylamide gels, and transferred to nitrocellulose membranes. The region containing the polypeptides were cut out, solubilized in dimethyl sulfoxide, mixed with Freund's adjuvant, and injected at 2-wk intervals into a male rabbit. The antiserum used in this study was obtained 8 wk after the initial injection and crossreacted with antigens on Western blots of uterine flushings and uterine culture medium obtained from ovariectomized estradiol (E2)-primed cats treated with P, and from pregnant preimplantation animals. Polypeptides in three molecular weight regions crossreacted with the antisera (Mr approximately equal to 28,000, pI 5.5 6.0; Mr approximately equal to 36,000, pI 6.0 6.5; Mr approximately equal to 41,000, pI 5.5 6.0), and each region consisted of several isoelectric variants. The Mr approximately equal to 28,000 proteins were the dominant form observed in culture media, and the Mr approximately equal to 36,000 proteins were the major form present in uterine flushings. The antigens were not detected in uterine flushings or culture medium obtained from ovariectomized, E2-treated, and estrous animals. The antigens were also absent in serum and other reproductive and nonreproductive tract tissues. Immunocytochemical analysis demonstrated that antigen staining was limited to the epithelial cells of the deeper uterine glands of the P-dominated animal. Immunoperoxidase staining was diffuse throughout the cytoplasm of these epithelial cells. Thus, the epithelial cells of the uterine glands in the P-dominated cat synthesize and secrete a complex group of P-dependent, uterine-specific proteins that may have potential functional significance during early blastocyst development and implantation.