The protein profile of each tissue depends on the expression of genes that are regulated, among others, by sex steroids. Comparing the profiles of sexually immature and mature females should therefore define markers of age-related changes. The aim of this study is to compare the pattern of proteins in the endometrium of heifers and pre-pubertal female calves by using difference in gel electrophoresis (DIGE), and to identify the presence and amount of similar or significantly different proteins. Endometrial samples were collected in slaughterhouse from heifers aged between 14-27 months (n = 6; sexually mature) and calves between 0.5-2 months (n = 6; sexually immature). All animals were healthy, Holstein-Friesian bred animals. Samples were subjected to fluorescent staining and 2D electrophoresis. Out of more than 900 spots detected in the endometrium of heifers, 73% were similar to calves. Selected spots were identified. Angiopoietin-2 and dynamin-like protein were detected only in heifers. These proteins are involved in angiogenesis and cell membrane remodelling, respectively. Aldose reductase, and phospholipase, which are important for prostaglandin metabolism, were present in different amounts in both sources. These results help to further understand the mechanism of steroid hormone action and look for markers of bovine endometrium status. Moreover, fluorescent staining appeared to be an useful tool when comparing 2 samples from different sources.