The putative antidepressant drug tiflucarbine (BAY P 4495) has previously been shown to inhibit calmodulin-dependent cyclic nucleotide phosphodiesterase competitively with respect to calmodulin. In order to determine whether this effect is mediated by a direct interaction with calmodulin, we measured the effects of radiolabelled triflucarbine in a direct ligand binding assay, using agarose-immobilized calmodulin. [3H]Tiflucarbine associated with low micromolar affinity with an apparently homogeneous class of recognition sites on calmodulin-agarose. No binding could be observed on calmodulin-deficient agarose. The effect was specific, saturable and reversible. Triflucarbine was the most potent calmodulin antagonist from a variety of structural analogues examined. The potencies of these derivatives to inhibit calmodulin-stimulated phosphodiesterase significantly correlated with their affinities towards the tiflucarbine binding site on calmodulin. No such correlation was evident when structurally unrelated reference compounds were tested. The association of tiflucarbine with calmodulin thus appears pharmacologically specific and selective and possibly contributes to the potent antidepressant activity of the drug.