The guanine nucleotide-binding proteins which mediate hormonal inhibition of adenylate cyclase as well as hormonal regulation of other membrane functions are α, β, and γ heterotrimers which are structurally homologous to each other. In brain, the predominant guanine nucleotide-binding component is a 39-kDa protein whose physiological role is as yet unknown. We have used N-ethylmaleimide to define functionally important sulfhydryl groups on α39. Three cysteine residues in the molecule are reactive in unliganded α39. Alkylation of two of these is reduced when guanosine 5'-(3'-O-thio)triphosphate (GTPγS) is bound. We have isolated and sequenced tryptic peptides containing the three reactive cysteines. The octapeptide containing the GTPγS-insensitive cysteine is at a position equivalent to amino acids 106-113 of the transducin α subunit (Lochrie, M. A., Hurley, J. B., and Simon, M. I. (1985) Science 228, 96-99). However, the equivalent peptide in transducin does not contain a cysteine residue. Alkylation of this cysteine blocks ADP-ribosylation of cysteine 351 by pertussis toxin. However, alkylation does not prevent association of α with the β · γ subunits nor does it inhibit GTPase activity. The two GTPγS-sensitive cysteines are at positions equivalent to cysteines 139 and 286 of the transducin α subunit. Alkylation of these residues inhibits GTPase activity. Neither of these GTPγS-sensitive cysteines are in those regions of α39 which are highly homologous to the GTP-binding site of elongation factor Tu (Jurnak, F. (1985) Science 230, 32-36). However, both are present in the brain 41-kDa guanine nucleotide-binding protein and in the two transducins. The conservation of these cysteine residues suggests that they are important for the function of the subunits.