The 5'-nucleotidase located in the cytoplasmic fraction of bovine brain cortex was purified to electrophoretic homogeneity. The molecular weight was 134,000 daltons in the presence of sodium deoxycholate, whereas the enzyme formed high molecular weight aggregates in the absence of detergent. The purified enzyme showed the same kinetic and electrophoretic behaviour as the enzyme present in the original cytoplasmic fraction, and the presence of surfactants did not change the Km and Vm values. The nucleotidase from this source was a phosphohydrolase of 5'-mononucleotides acting on the deoxyribonucleotides and ribonucleotides of purines and pyrimidines. 5'-IMP was the preferred substrate; the optimum pH was 7.5. The study of the influence of the temperature on the initial reaction rates allowed calculation of the delta Ea and delta H degrees values. The variation of Vm and Km with a change in pH suggests the existence of a sulfhydryl group and an imidazole group in the enzyme-substrate complex.