The distribution of haloperidol-sensitive (+)[3H]N-allylnormetazocine ((+)[3H]SKF-10,047) binding sites (sigma sites) in subcellular fractions of rat brain homogenates was extensively characterized. In synaptosomal fractions, enriched in choline acetyltransferase activity, sigma sites were present in lower concentrations than in whole brain homogenates. On the other hand, microsomal and myelin fractions were found to be enriched in sigma sites. A similar pattern of enrichment was seen for 5'-nucleotidase activity, a general plasma membrane marker. However, subsequent experiments in which microsomes were subfractionated on linear sucrose gradients led to the recovery of sigma sites over a significantly lower density range than 5'-nucleotidase activity or ATP-stimulated [3H]ouabain binding, an additional plasma membrane marker. In addition, previously reported distributions of a number of other subcellular markers, including those for endoplasmic reticulum, were found to contrast with the observed distribution of sigma sites. It is concluded that rat brain sigma sites are not concentrated at synaptic regions of plasma membrane. However, the possibility that sigma sites are localized to specialized areas of nonsynaptic plasma membrane cannot be excluded.