1. The contribution of target cells in controlling the functional properties of sympathetic neurons was investigated using pure neuronal cultures and co-cultures of neurons with their physiological target cells. 2. Chick embryo sympathetic neurons cultured alone exhibited maximal elevation of cytosolic free Ca2+ ([Ca2+]i) and release of tritiated noradrenaline ([3H]NA) when given ten stimulating pulses at 1 Hz but not at 10 Hz, yielding a negative frequency-release response. Stimulation-evoked release was only slightly enhanced by the K+ channel blocker tetraethylammonium (TEA, 10 mM). 3. When sympathetic neurons were co-cultured with cardiac cells of the chick embryo, electrically stimulated transmitter release and neuronal [Ca2+]i were reduced by 3- to 5-fold. Co-cultured neurons had a positive stimulation frequency--[3H]NA release response and 5- to 7-fold facilitation of release by TEA. 4. Voltage-clamped Ca2+ current density was decreased from 0.61 +/- 0.13 pA micron-2 in neurons alone to 0.19 +/- 0.03 pA micron-2 in co-cultured neurons. 5. Neonatal rat superior cervical ganglion (SCG) neurons were also relatively insensitive to TEA when cultured alone, but [3H]NA release was greatly facilitated by TEA when tested in SCG neurons co-cultured with rat neonatal cardiac myocytes. 6. The cardiac cell-induced changes in Ca2+ handling and release properties were produced within 24 h by sympathetic neuroeffector cells, but not by skeletal muscle cells or sensory neurons, and did not occur spontaneously in neurons grown alone for up to 6 days. 7. The frequency and TEA responses of neurons grown with cardiac cells are characteristic of responses seen in sympathetic neuroeffector organs. We conclude that physiological targets play a crucial role in development of normal transmitter-release properties by controlling Ca2+ homeostasis in sympathetic neurons.