Zymogram analysis using isoelectric focusing on polyacrylamide gels was performed to characterize and distinguish two Toxoplasma gondii isolates ("strains" BK and RH). The activity of the following 14 enzymes in the cell lysates was investigated: IDH, MDH, ME, 6PG, G6P, LDH, IPO, HEX, PGM, EST, ALP, ACP, LAP, and PGI. Nine enzymes (IDH, G6P, LDH, HEX, PGM, EST, ALP, ACP, and PGI) showed distinct and reproducible banding patterns, and four of them (IDH, G6P, EST, PGI) enabled a reliable distinction of the two Toxoplasma gondii isolates. A contamination of the parasite extracts with host proteins could be excluded by comparison of the enzyme activities of the Toxoplasma isolates with mouse peritoneal exudate cells. Isoenzyme analysis proved to be a helpful method for a characterization and a distinction of Toxoplasma gondii isolates.