Following up the finding that the non-imidazole drug clozapine shows a considerable histamine H3 receptor antagonistic activity, a series of analogues and metabolites (clozapine-N-oxide, and N-desmethylclozapine) were tested for their affinity towards the H3 receptor using the radiolabelled H3 antagonist [125I]-iodophenpropit. Qualitative structure affinity relationships are derived for the tested compounds. In the clozapine molecule four structurally different moieties may be considered. In comparison with the affinity for the H3 receptor shown by clozapine, the following main conclusions can be drawn: The 4-piperazinyl region does not allow substituents longer than a CH3 or electronegative atoms such as an O (as in clozapine-N-oxide); the lack of the CH3 group (as in N-desmethylclozapine) also reduces the affinity for H3 receptors. Substitutions at the 5-diazepine position do not drastically alter the affinity for the H3 receptor, although a basic nitrogen is favoured over CH2, O, or S. The 8 position in ring I is an important modulatory site for H3 affinity; electronegative substituents such as chloro and fluoro in this aromatic ring increase the affinity. When these substituents are, however, present at position X2 in the ring, they disable binding to the H3 receptor. The two major clozapine metabolites (clozapine-N-oxide, and N-desmethylclozapine) will not be responsible for a possible contribution of the H3 receptor antagonism to the clinical profile of clozapine.