Norepinephrine (NE)-producing cells of the nucleus locus ceruleus and dopamine (DA)-producing cells of the substantia nigra were dissected microscopically from embryonic rat brain, explanted, and maintained in culture for up to 5 weeks. The cultured neurons of both brain regions showed normal maturation of axons and dendrites and formed ultrastructurally defined synaptic contacts. Fluorescence microscopy of cultured neurons from both brain regions showed typical in situ cytological features: long axonal processes with multiple varicosities for locus ceruleus cultures, and smooth, wispy nonvaricose processes in the substantia nigra cultures. All cultures processed for fluorescence microscopy contained specific catecholamine-fluorescent cells. By radioenzyme assay for catecholamines, more than half of the locus ceruleus cultures contained measurable (>10 pg) quantities of NE and DA, but, unlike results on intact brains, DA content exceeded NE content. Cultures of substantia nigra neurons retained no NE and very little DA. Media from substantia nigra and locus ceruleus cultures contained substantial quantities of DA. Addition of reserpine (10 muM) to the medium depleted locus ceruleus neurons of both amines. The long survival time in culture of locus ceruleus cells, the normal appearance of fluorescent cell bodies and processes, the apparent development of morphologically specialized interneuronal connections, and the ability to synthesize and store NE make these cultures ideally suited for neurophysiological recording as well as morphological, biochemical, and pharmacological experiments.