We examined the effects of N-methyl-L-serine (NMS), an amino acid derivative, on hyaluronan (HA) synthesis in human skin fibroblasts. NMS (1-10 mM), but not L-serine, stimulated the incorporation of [(3)H]glucosamine into HA dose-dependently, with a maximum stimulation of 1.5-fold compared to the control. The effect of NMS was specific for HA production, because there was no change in sulfated glycosaminoglycan formation. Neither the N-methyl derivatives of L-glycine or L-alanine, nor N-methyl-D-serine, could stimulate HA synthesis, indicating that the beta-hydroxyl group and the L-configuration were essential for the activity. Gel filtration of the products showed that NMS stimulated the production of high-molecular-mass HA (>10(6) D) without affecting the production of low-molecular-mass HA. NMS required 24 h to stimulate HA production, and when fibroblasts were pretreated for 10-24 h with NMS (1-10 mM), membrane-associated HA synthase activity was increased dose-dependently. Thus, a second messenger is likely to be involved in the stimulation of HA production by NMS.