Copper is a divalent cation with physiological importance since deficiency of copper homeostasis can cause serious neurological diseases. ATP is an important signalling molecule stored at nerve endings and its inactivation is promoted by ecto-nucleotidases. In this study, we verified the effect of acute and subchronic copper treatments on ecto-nucleotidase activities in zebrafish brain membranes. Treatment with copper sulfate (15 microg/L) during 24h inhibited ATP hydrolysis (16%), whereas ADP and AMP hydrolysis were not altered. Nevertheless, a 96-h exposure with the copper concentration mentioned above inhibited NTPDase (31% and 42% for ATP and ADP hydrolysis, respectively) and ecto-5'-nucleotidase (40%) activities. NTPDase1, NTPDase2_mg and NTPDase2_mv transcripts were decreased after copper exposures during 24 and 96 h. Subchronic copper treatment also reduced the NTPDase2_mq and ecto-5'-nucleotidase expression. In vitro assays demonstrated that NTPDase activities were reduced after copper exposure during 40 min. ATP hydrolysis was inhibited at 0.25, 0.5 and 1mM (13%, 31% and 48%, respectively) and ADP hydrolysis also had a significant decrease at these same copper concentrations (41%, 63% and 68%, respectively). In contrast to the subchronic exposure, no significant changes on ecto-5'-nucleotidase were observed after in vitro assays. Lineweaver-Burk plots suggested that both inhibitory effects on nucleotide hydrolysis may occur in a non-competitive manner. Altogether, these findings indicate that copper is able to promote distinct changes on ecto-nucleotidases after in vivo and in vitro treatments and, consequently, it could control the nucleotide and nucleoside levels, modulating the purinergic signalling.