Cholera toxin is known to inhibit lymphocyte activation in vitro, an effect attributed to its ability to activate adenylate cyclase and increase intracellular cyclic adenosine monophosphate. In these studies the effects of both cholera toxin (CT) and its purified binding subunit (CT-B) on lymphocyte proliferation in vitro was examined, using a variety of cell activators. We found that both CT and CT-B inhibited mitogen- and antigen-induced T cell proliferation and anti-IgM-induced B cell proliferation in a dose-dependent manner. However, only CT-inhibited lipopolysaccharide-induced B cell proliferation. Neither CT nor CT-B inhibited antigen uptake and presentation by macrophages. The CT-B preparation used was shown not to activate lymphocyte adenylate cyclase, although CT itself was a strong activator of this enzyme. Both molecules had to bind to the lymphocyte surface in order to inhibit. The time course of inhibition of both CT and CT-B was similar in that either could be added up to 24 hr after culture initiation and still inhibit substantially. The addition of excess human recombinant interleukin 2 to the cultures did not affect the inhibition by CT, and had only a partial affect on inhibition by CT-B. Similarly, CT was able to substantially inhibit recombinant interleukin 2-dependent T lymphoblast proliferation, whereas CT-B had only a small inhibitory effect. Inhibition was not major histocompatibility complex-restricted. We conclude that the binding of CT or CT-B to the lymphocyte surface membrane interferes in some way with the activation mechanism leading to proliferation. The inhibition mediated by CT-B does not involve the stimulation of intracellular adenylate cyclase. CT appears to inhibit both by binding via its B subunit and by activation of adenylate cyclase via its A subunit.