Two highly selective radiolabeled probes for sigma receptors were found to bind with high affinity and capacity to membranes from undifferentiated PC12 cells. [3H]1,3-di-o-tolylguanidine [( 3H]DTG) bound with Kd = 23.7 +/- 4.6 nM and Bmax = 2025 +/- 660 fmol/mg protein. The Kd and Bmax for [3H](+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([3H](+)-3-PPP) were 86.3 +/- 21.6 nM and 1539 +/- 242 fmol/mg protein, respectively. These binding parameters were comparable to those observed in guinea pig brain, although the Kd for [3H](+)-3-PPP was 3-fold higher in the PC12 membranes. Both the PC12 and guinea pig brain sites exhibited high affinity for haloperidol, moderate affinity for phencyclidine (PCP), and negligible affinity for MK-801, apomorphine, and (-)-sulpiride. These data suggest a relationship of the PC12 site to sigma receptors. However, all (+)-opiates [+)-benzomorphans and (+)-morphinans) tested bound with markedly lower affinity to the PC12 site compared to guinea pig brain. These include (+)-N-allylnormetazocine [+)-SKF 10,047), (+)-pentazocine, and dextrallorphan. In fact, PC12 sites exhibited preference for (-)-benzomorphans, the reverse stereoselectivity of guinea pig brain sites. Binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) could not be detected, demonstrating absence of PCP receptors on this cell line. Differentiation of cells by treatment with nerve growth factor had no effect on sigma binding parameters. Membranes from guinea pig brain and PC12 cells were photoaffinity-labeled using [3H]azido-di-o-tolylguanidine. In guinea pig brain, a polypeptide of 25 kDa was specifically labeled. However, label was incorporated into polypeptides of 18 kDa and 21 kDa in membranes from PC12 cells. In view of the otherwise similar binding characteristics, the marked differences in affinity for (+)-benzomorphans and molecular weight suggest that PC12 cells contain a molecular form of sigma receptor distinct from that predominant in guinea pig brain. This raises the possibility of multiple sigma receptor types.