1. We performed single channel analysis on human alpha3 acetylcholine receptors (AChRs) in Xenopus oocytes and native AChRs from the human neuroblastoma cell line IMR-32. alpha3 AChRs exhibit channel properties that reflect subunit composition. 2. alpha3beta2 AChR open times were 0.71 +/- 0.14 and 3.5 +/- 0.4 ms with a predominant conductance of 26 pS. alpha3beta4 AChRs had open times of 1.4 +/- 0.2 and 6.5 +/- 0.8 ms and a predominant conductance of 31 pS. Burst times were 0.82 +/- 0.12 and 5.3 +/- 0.7 ms for alpha3beta2 and 1.7 +/- 0.1 and 16 +/- 1 ms for alpha3beta4. Desensitization was faster for AChRs with the beta2 subunit than for those with the beta4 subunit. 3. One open time for alpha3alpha5beta2 AChRs (5.5 +/- 0.3 ms) was different from those of alpha3beta2 AChRs. For alpha3alpha5beta4 AChRs, an additional conductance, open time and burst time (36 pS, 22 +/- 3 ms and 43 +/- 4 ms, respectively) were different from those for alpha3beta4 AChRs. 4. alpha3 AChRs were inhibited by hexamethonium or mecamylamine. The rate constants for block of alpha3beta4 by hexamethonium and of alpha3beta2 by mecamylamine were 1.2 x 107 and 4.6 x 107 M-1 s-1, respectively. 5. AChRs from IMR-32 cells had a predominant conductance of 32 pS and open times of 1.5 +/- 0.3 and 9.6 +/- 1.2 ms. These properties were most similar to those of alpha3beta4 AChRs expressed in oocytes. Antibodies revealed that 5 +/- 2 % of IMR-32 alpha3 AChRs contained alpha5 subunits and 6 +/- 2 % contained beta2 subunits. IMR-32 alpha3 AChRs are primarily alpha3beta4 AChRs.