Babesia bigemina merozoites enter their host's erythrocytes by an unknown mechanism that likely involves parasite surface components. Identification of the parasite ligands involved in invasion is hampered by a lack of basic information about the invasion characteristics of Babesia bigemina. Therefore, restrictions on the species of red blood cells (RBC) that are susceptible to invasion were examined as well as the roles of erythrocyte ligands. An invasion assay and a proliferation assay were developed for this study. Unlike some other species of Babesia that infect cattle, B. bigemina failed to enter RBC from most animals that are not natural hosts, suggesting that a species restricted receptor mechanism mediates invasion. Two carbohydrates which are prominent on the surface of bovine erythrocytes, N-acetylglucosamine and N-acetylgalactosamine, when added to cultures, reduced the ability of B. bigemina merozoites to invade erythrocytes. Neuraminidase or trypsin treatment of bovine erythrocytes significantly decreased their susceptibility to invasion whereas chymotrypsin had little effect. These data imply that proteinaceous erythrocyte ligands and carbohydrate residues may be involved in the invasion process. Identification of a species-specific pattern of invasion and RBC treatments that render cells refractory to invasion may provide the basis for the characterization of B. bigemina erythrocyte binding molecules based on their differential binding to invasion competent and refractory cells.