Subjecting either P2 fraction or purified synaptosomes isolated from rat brain to periods of anoxic incubation at 30 degrees resulted in activation of dopamine synthesis from tyrosine. This activation was approximately 2.5-fold when the anoxic incubation was carried out at pH 6.2 but was not significant when the pH was 7.4. Measurements of the tyrosine hydroxylase activity at pH 6.2 in Triton X-100-treated preparations of the P2 fraction showed that, after 20 min of anaerobiosis, the Km for pterine cofactor decreased by 39% and the Ki for dopamine increased by 44%; there was no change in the Km for tyrosine. Half-maximal activation of dopamine synthesis occurred in 10 min of anaerobic incubation, and the reversal upon addition of oxygen had a half-time of 15 min. Addition of forskolin or dibutyryl cyclic AMP to anaerobic incubations of P2 fraction did not result in significant activation of dopamine synthesis. Either the removal of calcium or the addition of calmodulin inhibitor, trifluoperazine, substantially decreased the activation of dopamine synthesis induced by periods of anaerobiosis. It appears that during anoxic incubation tyrosine hydroxylase underwent an activation which occurred over a period of minutes, was stable to detergent treatment, and was fully reversed over a period of minutes following reoxygenation. This activation was, at least in part, dependent on the presence of calcium and was sensitive to the calmodulin antagonist trifluoperazine.