In vivo microdialysis was used to compare the effects of β-endorphin upon dopamine (DA) release in the nucleus accumbens (NAC) of anesthetized versus freely moving rats, and to examine the role of the the mesolimbic DA system in mediating both the motoric and secondary reinforcing effects of this peptide. Microdialysis probes were inserted into the NAC and perfusates were analyzed for DA and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), using a reversed phase HPLC system with electrochemical detection for separation and quantification. Intracerebroventricular (ICV) administration of β-endorphin (2.5 and 5.0 µg) increased DA release and metabolites in both freely moving and anesthetized rats. This effect was of greater magnitude and duration in freely moving rats and was accompanied by stimulation of locomotor activity. The 5 µg dose also functioned as a secondary reinforcer in a conditioned place preference paradigm. A higher dose of β-endorphin (7.5 µg) stimulated DA release and metabolites in anesthetized rats but failed to affect these parameters in freely moving rats. At this dose, catalepsy and a loss of the reinforcing effects of this peptide were observed. These data demonstrate marked differences in the effects of β-endorphin upon DA release in the awake versus anesthetized rat. Further, the finding that the reinforcing and locomotor stimulating effects of β-endorphin only occur at those doses which stimulate DA release suggest that this action is critical for the expression of both behavioral effects.