Rabbit aortic smooth muscle cells cultured for 14 days in second passage produce significant amounts of protein-bound radioactive Nt-methylhistidine when pulsed with radiolabelled histidine. Much of the radioactive Nt-methylhistidine is located in the actin synthesized by these cells. Pulse chase data also suggest this newly synthesized Nt-methylhistidine disappears from the cell layer at two distinctly different rates: one with a half-life of 2.4 days, and the other with a half-life of approximately 17 days. Radioactive Nt-methylhistidine to histidine ratio reaches a maximum approximately 8 days following the 1-day [14C]histidine pulse. During the initial 15-day chase period, there is a substantial net accumulation of total Nt-methylhistidine in the culture flasks. The data presented in this communication indicate that methods to evaluate Nt-methylhistidine synthesis and disappearance in cultures are feasible when radiolabelled histidine is employed.