The stability of sodium 5,6-benzylidene-L-ascorbate (SBA), consisting of two diastereomers, was investigated by high-performance liquid chromatography. In extensively acidic buffer, the acetal in SBA was immediately cleaved to liberate ascorbic acid and benzaldehyde. At higher pH, the cleavage of SBA was significantly reduced, but the degradation (due to possible opening of the lactone ring) of SBA was stimulated. The degradation rate of SBA was significantly increased with increasing temperature, and was higher than that of ascorbic acid or benzaldehyde. SBA was degraded with incubation time in culture medium, with accompanying loss of its biological activity, but only a marginal concentration of benzaldehyde, but not of ascorbic acid, was produced from SBA. The amount of SBA extracted from the apoptosing leukemic cells by 70% acetonitrile amounted to about 0.04% of that present in the medium fraction. These data suggest that SBA itself, but not contaminating ascorbic acid nor benzaldehyde, is responsible for the antitumor activity of SBA.