In this paper, by means of a push-pull perfusion technique, we have examined the local effect of ovine prolactin (oPRL) on the spontaneous in vivo dopaminergic (DAergic) activity of the nucleus accumbens (NAcc), substantia nigra (SN) and ventral tegmental area (VTA) of living male rats. First, we found a 2-fold spontaneous rise of 3,4-dihydroxyphenylacetic acid (DOPAC) output during the afternoon hours of the photoperiod only in the NAcc. In addition, native oPRL (10 ng/µl for 20 min), but not boiled oPRL, locally perfused into the NAcc caused a significant elevation in DOPAC output without changes in homovanillic acid or 5-hydroxyindoleacetic acid. In a similar preparation, male rats bearing push-pull cannulae in the SN or VTA were infused locally with oPRL (10 or 50 ng/µl) for 20 min. The hormone did not alter the efflux of DOPAC from these DA cell body/dendrite areas. These results indicate that oPRL can activate the mesolimbic DA system through local actions on DA presynaptic terminals of the NAcc without affecting the SN or VTA.