In order to explore the feasibility of producing a Babesia divergens live vaccine free of bovine material contaminants the parasite's ability to grow in human, sheep and horse erythrocytes and serum and serum-free medium was investigated. B. divergens was successfully maintained in bovine erythrocytes overlaid with serum-free HL-1 medium. Supplementation of the culture medium with bovine or sheep serum improved parasite growth (monitored by measuring parasitaemia and uptake of tritiated hypoxanthine) whereas horse and human sera reduced parasite growth. As assessed by Giemsa's stained and FITC-labelled blood smears, the parasite invaded all erythrocyte types. Polyparasitism was less common in sheep and horse erythrocytes than in bovine and human erythrocytes. Accole stages were observed in bovine, human and sheep but not in horse erythrocytes. Proliferation following invasion was higher in human but lower in horse and sheep erythrocytes compared with bovine erythrocytes. Long-term cultures of B. divergens reached similar peak parasitaemias in human, sheep and bovine erythrocytes. Attempts to establish long-term cultures in horse erythrocytes failed. These results suggest that B. divergens is not host specific at the level of host cell attachment and invasion. Instead, parasite survival appears to be decided once the organism has gained access into the cell.