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Total protein secreted in the intrauterine lumen increases between day 10 and 13 post-estrus in both cyclic and pregnant gilts. The objective of this experiment was to identify those intrauterine proteins whose secretion changes during this time period. Sixteen mature gilts were either mated (day 0) or remained cyclic and were slaughtered at either day 10 or day 13 (n = 4 per status by day). At slaughter, each uterine horn was flushed with 20 ml Minimal Essential Medium. Flushings were dialyzed extensively against distilled water. A 0.5 ml aliquot of each was lyophilized, subjected to two-dimensional PAGE, and protein spots were identified following Coomassie staining of each gel. Densitometry was used to compare relative amounts of each spot. After statistical analysis, spots that differed due to either day, status, or day by status interaction were excised and digested in-gel with trypsin. The resulting peptides were analyzed by tandem mass spectrometry (MS/MS). Using MS/MS data, protein identification for each spot was attempted. There were 280 matching spots, of which 132 were significantly (P < 0.05 or 0.01) affected by pregnancy status, day, or the day by status interaction. Most (73%) spots increased from day 10 to day 13 with no effect of pregnancy. Several spots were identified as proteases or their inhibitors. Others potentially modify glycolipids and/or glycoproteins. These results indicate that the concentrations of many proteins within the intrauterine environment during early pregnancy are independent of the conceptus and could play roles in regulating the endometrial or conceptus glycocalyx.