The aim of this study was to examine behavioral and biochemical effects of nafadotride, the new dopamine D3 receptor antagonist, and to compare it with haloperidol (dopamine D2 receptor antagonist) and clozapine (predominate dopamine D4 receptor antagonist). Each drug was injected to adult male Wistar rats intraperitoneally, each at a single dose and for 14 consecutive days. Thirty minutes after single or last injection of the examined drugs, the following behavioral parameters were recorded: yawning, oral activity, locomotion, exploratory activity, catalepsy and coordination ability. By HPLC/ED methods, we determined the effects of the examined antagonists on the levels of biogenic amines in striatum and hippocampus: dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and noradrenaline (NA). Additionally, DA and 5-HT synthesis rate was determined in striatum and 5-HT in hippocampus. The results of the study indicate that nafadotride, the dopamine D3 receptor antagonist, has a behavioral and biochemical profile of action different from that of haloperidol but partially similar to that of clozapine.