In cattle, successful recognition of pregnancy depends on a biochemical interplay between endometrial and conceptus secretions. Because failure of pregnancy recognition accounts for 30-40% gestation losses, understanding mechanisms regulating this event may lead to increasing profits in the cattle industry. The goal of the present study was to characterize the protein profile of uterine washings collected from cyclic and pregnant cows during the critical period for pregnancy recognition. In Experiment 1, uterine flushings from cyclic (n = 15) and inseminated cows (n = 30) were collected in vivo, through a Foley catheter, on Days 14, 16, or 18 post-estrus. Protein composition of washings was evaluated by SDS-PAGE, coomassie staining, and densitometry. Overall, there was no effect of reproductive status or day post-estrus on optical density of protein bands in washings (P > 0.10). In Experiment 2, uterine flushings from cyclic (n = 12) and inseminated cows (n = 36) on Day 17 post-ovulation (i.e., Day 18 post-estrus) were collected either in vivo or post-mortem. Protein composition of washings was evaluated as in Experiment 1. Of the 20 protein bands analyzed, 5 had higher and 7 had lower optical densities in washings obtained in vivo in comparison to washings obtained post-mortem (P < 0.05). Optical densities of protein bands were similar between reproductive statuses (P > 0.10). In conclusion, sensitivity of analytical techniques was not sufficient to discriminate temporal nor reproductive status-specific proteins, neither in in vivo nor post-mortem flushings. Moreover, protein composition of washings depended on the method used for collection. It is speculated that a high relative abundance of serum proteins in washings diluted conceptus- and uterine-specific proteins to concentrations below the sensitivity of the methods used for protein detection.