While a number of studies have compared replication rates for vascular smooth muscle cells under different conditions, the absolute frequency of smooth muscle cell replication within the vessel wall has not been defined. This study reports a comparison of four parameters that might be expected to alter the accuracy of such counts. Neither autoradiograph exposure time, within specific time limits, nor dose of tritiated thymidine are major variables. Differences in absolute values obtained by autoradiography of enzyme-dispersed cells versus cells in cross-section, however, are reproducible. A comparison of frequency data from animals injected with tritiated thymidine during a 24-hour period compared with data from animals infused continuously for 1 week suggests that continuous infusion allows accumulation of labeled cells over time at a rate that is predictable from data of animals injected during 24 hours. In the current study it was found that although the frequency of tritiated thymidine-labeled thoracic aorta smooth muscle cells may vary according to the technique used in preparing the tissue, the daily rate of replication in a 4-month-old rat is estimated to be approximately 0.047% per day.