Ligand-gated nicotinic acetylcholine receptors (nAChRs) and purinergic ATP receptors are often expressed in the same peripheral and central neurons, and ATP and acetylcholine (ACh) are stored together in some synaptic vesicles. Evidence has suggested that nAChRs and ATP receptors are not independent and that some agonists strongly cross-activate and desensitize both receptor types. Rat sympathetic neurons and nAChRs expressed in Xenopus oocytes were studied to determine the significance of the interactions caused by the two agonist types. Current amplitudes included with separate or combined applications of ATP and nicotine are > 90% additive and independent. Half of all neurons tested responded to either ATP or nicotine but not to both, indicating differences in the expression of the two receptors. In neurons that expressed both receptors types, the nAChRs were inhibited by the activity-dependent open-channel blocker chlorisondamine. If the purinergic and nicotinic receptors were significantly dependent and coactivated, then blocking the ion channels opened by a nicotinic agonist should diminish the current activated by a purinergic agonist. That result was not seen; rather, complete open-channel block of nAChRS with chlorisondamine did not significantly alter the amplitude or kinetics of ATP-induced currents in the same neurons. Finally, when cloned nAChR subunits were expressed in oocytes, ATP activated only very small currents compared with the current activated by Ach. For the 13 different nAChR subunit combinations that were studied, ATP (50-500 microM) activated a current that ranged from 0 to 4% of the size of the current activated by 100 microM ACh. In summary, we find that there is little cross reactivity, and nAChRs and purinergic ATP receptors are predominantly independent, acting with separable physiological characteristics. Therefore the quantitative Ca2+ flux could be separately determined for nAChRs and ATP receptors. The fraction of total current that is carried by Ca2+ was quantitatively determined by simultaneously measuring the whole cell current and the associated change in intracellular Ca2+ with fura-2. For sympathetic neurons bathed in 2.5 mM Ca2+ at a holding potential of -50 mV, Ca2+ carries 4.8 +/- 0.3% (mean +/- SE) of the inward current through neuronal nAChRs and 6.5 +/- 0.1% of the current through purinergic ATP receptors. In conclusion, activity-dependent Ca2+ influx through predominately independent populations of nAChRS and ATP neurons can produce different intracellular signals at purinergic and cholinergic synapses.