Experiments were conducted to gain insight concerning the mechanism(s) whereby cerulenin and sodium butyrate affect chitin synthesis in Candida albicans. In vitro studies with isolated membrane-bound chitin synthase from C. albicans, strain 4918, showed that neither agent affected the level of either unactivated or trypsin-activated enzyme activity. Subsequent studies utilizing protoplasts revealed that early in the cell wall regeneration process, cells treated with cerulenin or butyrate synthesized chitin at a rate equal to untreated controls, as measured by the incorporation of [3H]-N-acetylglucosamine (GlcNAc) into acid-alkali insoluble material. However, after 40 min of incubation, the incorporation of [3H]GlcNAc into chitin is reduced in cells treated with either agent. On the other hand, samples taken during the same time intervals and analyzed by flow cytometry suggested that the amount of chitin synthesis in treated and untreated cells was identical. A marked decrease in fluorescence was observed in similar experiments using polyoxin D, a direct inhibitor of chitin synthase activity. Experiments that measured uptake of [3H]GlcNAc into both whole cells and protoplasts demonstrated that cerulenin and butyrate had no effect on the transport of the chitin precursor.