Concentrations of dopamine, serotonin, and some of their metabolites were analyzed by means of high-performance liquid chromatography in brain samples obtained from rats operantly conditioned to use one paw for water-reinforced lever pressing. In the first experiment, the side of paw usage was determined by physical constraint (forced-handedness condition), whereas in the second experiment the side of paw usage was not restricted (paw-preference condition). Differences in dopamine metabolism were detected between brain samples from the hemispheres located ipsi- and contralaterally to the side of paw usage. A higher dopamine metabolism (indicated by higher metabolite/transmitter ratios) was found in the amygdala ipsilateral to the paw used both under the forced-handedness and paw-preference condition. A higher level of dopamine in the contralateral septum was found in rats sacrificed immediately after 15 min of forced-handedness and an ipsilateral increase was found in rats analyzed 2 h after performance of this task. In addition, a higher dopamine metabolism in the ventral striatum, dorsal striatum, and amygdala was found in the forced-handedness and yoked controls groups than in rats analyzed 2 h after lever pressing. In the second experiment, rats in the paw preference group had a lower dopamine metabolism in the ventral and dorsal striatum, septum, and substantia nigra than did their yoked controls. These results show that changes in dopamine metabolism during conditioned lever pressing can be asymmetrical with respect to the side of paw usage, indicating that the dopamine neurons in the two brain hemispheres are asymmetrically involved in such behavioral tasks.