In membranes of S49 lymphoma cyc− variants, which lack a functional guanine nucleotide-binding component (Ns protein) mediating adenylate cyclase stimulation by hormones, guanine nucleotides decreased the forskolin-stimulated adenylate cyclase activity by maximally 40–60%. The potency order of the guanine nucleotides studied was guanosine 5′-[γ-thio]triphosphate(GTP[S])> guanosine 5′[β,γ-imido]triphosphate ≫ GTP > guanosine 5′-[β-thio]diphosphate (GDP[S]). GTP and GDP[S] acted as partial inhibitors; they competitively antagonized the GTP[S]-induced inhibition, which was half-maximal and maximal at about 3 nM and 100 nM GTP[S], respectively. Cholera toxin did not enhance the inhibitory action of GTP. The cyc− adenylate cyclase inhibition by GTP[S] occurred after a short lag period and persisted after washing the membranes. The inhibition was not affected by the pH (6.5–9.0) of the incubation medium and was largely independent of the concentrations of MgATP (up to 200 μM) and Mg2+ (up to 10 mM). In contrast, Mn2+ potently reduced the GTP[S]-induced cyc− adenylate cyclase inhibition. Similarly as observed with forskolin, GTP[S] decreased the cyc− adenylate cyclase activity stimulated by purified, preactivated Ns protein. Apart from Mn2+, inhibition of the cyc− adenylate cyclase by GTP[S] was prevented or reversed by various treatments, which have been shown to obliterate hormone-induced adenylate cyclase inhibition in other cell types, such as by N-ethylmaleimide, by limited proteolysis with trypsin and by a factor extracted from bovine sperm particles. The data indicate that in membranes of cyc− variants, which lack a functional Ns protein, a guanine nucleotide-binding component (Ni) is present, which mediates adenylate cyclase inhibition by guanine nucleotides. Such a component apparently also mediates adenylate cyclase inhibition by hormones in other cell types. Comparison of adenylate cyclase stimulation and inhibition by guanine nucleotides suggests that Ni is activated and inactivated by similar but not identical mechanisms as the Ns protein and that the relative activity states of these coupling components determine the activity of the adenylate cyclase.