The concentration of free and total homocysteine in plasma increases in time if blood is stored uncentrifuged after sampling. The increase is temperature dependent and the maximal increase in total plasma homocysteine at 37 degrees C was 3.0 mumol.L-1.h-1. Even at 4 degrees C there is a substantial increase, particularly of free plasma homocysteine. Plasma glutathione, cysteinylglycine, and gamma-glutamylcysteine also show an increase in time if whole blood is stored, whereas cysteine decreases. We show that the erythrocytes are responsible for most of the increase in plasma homocysteine and suggest that homocysteine is derived from adenosylmethionine-dependent protein carboxymethylations in the cells. We conclude that strict sampling conditions are necessary when plasma homocysteine and especially its free fraction are assayed.