In the hippocampal neuronal in vitro system of HT22 cells, we studied the effects of clozapine (Cloz) and its metabolites clozapine-N-oxide (Cloz-N-oxide) and norclozapine (Norcloz) on 5-HT transporter affinity (K(M)) and uptake (V(max)), MAO-B affinity (K(M)) and maximal velocity (V(max)), as well as on 5-HT(2) receptor affinity and density. Clinically relevant concentrations of Cloz (200 and 400 ng/ml) and its metabolites (100 and 200 ng/ml) were used for the examination of the effects after short-term (4 h) and long-term (24 h) incubation. Statistical evaluation revealed that a significantly lowered 5-HT transporter affinity (higher K(M)) was related to higher concentrations of Cloz and its metabolites. A significantly higher 5-HT transporter uptake was dependent on both high concentrations of drugs and an increased time of incubation. No significant influence of the investigated independent variables on MAO-B affinity could be demonstrated, whereas a significant drug-related increase of MAO-B velocity was detectable. Additionally, low and high concentrations of Cloz and its metabolites induced a higher 5-HT(2) receptor affinity (lower K(D)). No significant influences of the investigated independent variables on 5-HT(2) receptor density were detectable. The results of the present study show that Cloz and its metabolites induce significant alterations in serotoninergic parameters of hippocampal HT22 cells, validating the system of hippocampal HT22 cells for further examinations of the mechanisms of action of atypical neuroleptics.