The affinities of antagonists at histamine H1 receptors on HeLa cells have been determined from inhibition of histamine-induced inositol phosphate formation in intact and from inhibition of [3H]mepyramine binding to HeLa cell membranes. The dissociation constants of mepyramine and (+)-chlorpheniramine were similar to values for binding to H1 receptors in other mammalian tissues, but much lower than the values reported in an earlier study with [3H]mepyramine and HeLa cell membranes. Evidence is presented that under conditions employed in the earlier study the binding of [3H]mepyramine is largely to secondary, non-H1 receptor sites.