Animal cell cultures generally require a nutrient-rich medium supplemented with animal serum. Adult bovine serum contains a variety of nutrients including inorganic minerals, vitamins, salts, proteins and lipids as well as growth factors that promote animal cell growth. To evaluate the potential use of gender-specific bovine serum (GSBS) for cell culture, the biochemical properties of male serum (MS), female serum (FS) and castrated-male serum (CMS) were investigated. Overall, the chemical profile of GSBS was similar to that of bovine references except for glucose, creatine kinase, lactate dehydrogenase and potassium. FS showed elevated total protein and sodium concentrations compared to MS and CMS. Proteins present in MS, FS and CMS but absent in fetal bovine serum (FBS) were selected by two-dimensional gel electrophoresis and identified by peptide mass fingerprinting. Some of the identified proteins are known to be involved in immune responses and the others have unknown physiological roles. Moreover, it was found that some proteins such as alpha-2-macroglobulin appeared to be gender-specific with higher contents in FS. Insulin and testosterone was significantly higher in MS, and $17{eta}$-estradiol and estrone were higher in FS, as compared to the other sera. Taken together, the results indicate that each GSBS has a different ratio of components. Differences in serum constituents may affect cell cultures in a different manner and could be beneficial, depending on the specific aim of cell cultures.