The activities of the enzymes NTPDase (EC 184.108.40.206, apyrase, CD39) and 5′-nucleotidase (EC 220.127.116.11, CD73) were analyzed in platelets from rats submitted to demyelination by ethidium bromide (EB) and treated with interferon β (IFN-β). The following groups were studied: I — control (saline), II — (saline and IFN-β), III — (EB) and IV — (EB and IFN-β). After 7, 15 and 30 days, the animals (n = 7) were sacrificed and the platelets were separated by the method of Lunkes et al. [Lunkes, G., Lunkes D., Morsch, V., Mazzanti, C., Morsch, A., Miron, V., Schetinger, M.R.C., 2004. NTPDase and 5′-nucleotidase in rats alloxan- induced diabetes. Diabetes Research and Clinical Practice 65, 1-6]. NTPDase activity for ATP and ADP substrates was significantly lower in groups II and III after seven days, when compared to control (p < 0.001). At fifteen days, ATP hydrolysis was significantly lower in group III and IV and higher in group II (p < 0.001), while there was an activation of ADP hydrolysis in group II (p < 0.001), when compared with the control. 5′-nucleotidase activity was significantly higher in group IV (p < 0.001) after seven days, and lower in the groups III and IV (p < 0.001) after fifteen days in relation to the control. No significant differences were observed in NTPDase and 5′-nucleotidase activities after thirty days. In conclusion, our study demonstrated that the hydrolysis of adenine nucleotides is modified in platelets of rats demyelinated and treated with IFN-β.