The uterine protein milieu of cyclic and ovariectomized (ovx) cows treated with estradiol-17S (E-2) and progesterone (P) alone or in combination was studied by various biochemical methods. Uterine lumenal fluids (UF) were collected nonsurgically via a foley catheter. Sephacryl S-200 chromatography revealed protein peaks specific to UF from cyclic cows had distribution coefficients at ambient temperature of .27 ± .02, .32 ± .02, .65 ± .03, .73 ± .06, and .91 ± .04; peaks at 4C were .35, .41 ± .04, .82 ± .05, 99 ± .04, and 1.20 ± .09. UF analyzed by HPLC had uterine specific protein peaks eluting at 7.27 ± .32, 15.42 ± .39, 17.71 ± .14, 18.31 ± .13, and 20.91 ± .38 ml. Native polyacrylamide disc gel electrophoresis at pH 8.3 revealed differences in the frequency of appearance between plasma and UF for proteins with relative mobilities (R_A) of .033, .415, .517, .709, .780, and .894. Mean relative percent collectively of the .894 band and albumin was higher (P < .01) in UF than in plasma. UF were obtained from ovx cows administered E-2 and Palone or in combination by Silastic® implant (I) after each of three consecutive 18 day treatment periods. During the third treatment period, supplemental daily steroid injections in the same ratios as found in the I were given to augment I hormone contributions. Proteins unique to UF after the first treatment period had R_A of .648, 1.13, and 1.66. One cow (8 g P, 2 mg E-2) had a basic protein migrating 1.35 cm toward the cathode. Proteins unique to UF after the second treatment period had mobilities of .645, 1.15, 1.22, 1.40,1.60, and 1.68. Three cows receiving treatments including four 2 g P I had a basic protein migrating 1.4 ± .3 cm (X ± S.D.) toward the cathode. Two cows in these same treatments had an additional basic protein migrating .2 to .3 cm into the gel. Acidic proteins unique to UF after the third treatment period had R_A of .638, 1.15, 1.24, 1.40, 1.58, and 1.67. The appearance of proteins with R_A greater than 1.15 was associated with treatments including P except one cow receiving E-2 only, having bands at 1.15 and 1.20. A basic protein migrating 1.34 ± .19 cm (X ± S.D.) towards the cathode was observed in 11 of 18 cattle receiving treatments including P compared with none from cattle not receiving P. These data indicate that UF from cyclic cows contains small molecular weight uterine specific proteins and at least one large molecular weight protein. At least four acidic proteins and two basic proteins appear to be induced by treatments including P. / Ph. D.