The lumped constant (LC) for the α‐methyl‐l‐tryptophan method to convert the brain's uptake of labeled α‐methyl‐l‐tryptophan into the regional rate of serotonin synthesis was estimated. The method involved independently estimating the unidirectional uptake constant of the tracer (α‐[14C]methyl‐l‐tryptophan) to the tissue and the tracee (tryptophan) (with the addition of a radioactive compound) and calculating their ratio. The LC was estimated from logarithmically transformed data. Similar experiments were performed using rats treated with the drug probenecid, which blocks the efflux of 5‐hydroxyindoleacetic acid (a metabolite of serotonin) from the brain. The experiments using probenecid, corrected for the difference in the levels of plasma free tryptophan (increased in probenecid‐treated rats) relative to control experiments, gave an average LC for the rat brain of 0.46 ± 0.14 (mean ± SD). This value was not significantly different from the one obtained in controls (0.43 ± 0.13). In addition, the LC was also calculated using unidirectional uptake constants in the probenecid‐treated rats for α‐methyl‐l‐tryptophan and l‐tryptophan. This LC value was 0.39 ± 0.10. There was no significant difference between these three LC values. Thus, an average ± SD LC of 0.42 ± 0.07 for 28 brain structures investigated in this study was obtained. Statistically the LC obtained in different structures had a variability that could be accounted for by errors in measurements alone. In other words, dispersion in the LC values could be fully accounted for by chance alone. Data confirmed that the LC value did not change when the rate of serotonin synthesis was increased by probenecid treatment. We also showed that the rate of 5‐hydroxyindoleacetic acid accumulation in probenecid‐treated rats was 58 pmol g−1 min−1 (rat brain), which is about twice as much as reported by others for a normal rat. This difference could also be accounted for by the increase in the plasma level of free tryptophan in probenecid‐treated rats.