The whole blood from three human subjects, one dog and four rabbits was spiked with methotrexate (MTX) to yield appropriate concentrations, and was incubated at 37 degrees C and 50 oscillations per min. MTX was found to exhibit unusual distribution kinetics in whole blood; plasma MTX concentrations generally dropped to a minimum at about 5 min then increased and fluctuated somewhat irregularly afterwards. Differences of up to 21, 19 and 32% between maximum and minimum were found in the studies from human, dog and rabbit, respectively. These unusual distribution phenomena might be due to the Schiff base formation between free primary amino group(s) of MTX and free fatty aldehyde groups on blood cell membrane. Similar unusual distribution kinetics were observed with blood from three human subjects after kept at room temperature or in the refrigerator, and blood collected from three dogs after intravenous bolus or infusion of MTX. Formation of three metabolites including 7-hydroxymethotrexate was only found in blood from rabbits, but not from humans and dogs.