A new method for estimation of in vitro neurosecretory activity of tuberoinfundibular dopaminergic (TIDA) neurons was developed by measuring the rate of synthesis of dihydroxyphenylalanine (DOPA) in the median eminence of hypothalamic slices. Sagittal hypothalamic slices of ovariectomized rats were incubated in a medium containing 3-hydroxybenzylhydrazine (NSD 1015), an inhibitor of DOPA decarboxylase. DOPA accumulated in the median eminence following incubation with NSD 1015 was determined by high-performance liquid chromatography with electrochemical detection. The amount of DOPA accumulated in vitro in the median eminence was maximal in a medium containing 10 mM NSD 1015 and linear up to 120 min at 37°C. Increasing the concentration of tyrosinein medium stimulated the synthesis of DOPA in the median eminence. The synthesis of DOPA was blocked by 1 mM α-methyltyrosine, an inhibitor of tyrosine hydroxylase. The rate of in vitro synthesis of DOPA in the median eminence was 33% of that of in vivo synthesis. Incubation in a medium containing 50 mM K<sup>+</sup> to depolarize neurons caused a 2.4-fold increase in DOPA synthesis in the median eminence. The high K<sup>+</sup>-induced increase in DOPA synthesis was blocked by omission of Ca<sup>2+</sup> and addition of 1 mM EGTA into the medium, suggesting Ca<sup>2+</sup> dependency of depolarization-activated DOPA synthesis. These results indicate that this in vitro assay is a useful means to study the regulatory mechanisms of TIDA neurons.