A polyclonal rabbit antibody (9120) against the alpha subunit of the "other" guanine nucleotide-binding protein Go (alpha o) was raised against a synthetic alpha o peptide fragment (Asp-Gly-Ile-Ser-Ala-Ala-Lys-Asp-Val) attached to a branched core system. Antiserum 9120, at a final dilution of 1:400, can detect alpha o in as little as 0.2 microgram of Go on immunoblots, and, at a final dilution of 1:20,000, can detect alpha o in 1 microgram of Go on immunoblots. Antiserum and affinity-purified antibody are specific to alpha o. No cross-reactivity was detected to the alpha subunits of the stimulatory or inhibitory guanine nucleotide-binding regulatory proteins or of transducin (alpha s, alpha i, or alpha T) or to the beta or gamma subunits. Immunoblots revealed a high density of alpha o in rat brain and lung membrane preparations, but other tissues (such as adipose tissue, heart, erythrocytes, and liver) have no detectable alpha o. Developmental studies showed that alpha o in rat brain was low before birth, increased after birth, and reached the full adult level at 4 weeks of age. In contrast, ADP-ribosylation of 40-kDa proteins increased for up to 1 week and then decreased. Immunocytochemistry revealed that alpha o was localized to somatic and synaptic membranes in rat brain, whereas little or no alpha o was detected in the cytoplasm of neuronal cell bodies. Our observations suggest that Go in brain might have a role in membrane signal transduction at synaptic and extrasynaptic sites.