The 5'(3')-nucleotidase earlier partially purified from rat liver by Fritzson and Smith [1971) Biochim. Biophys. Acta 235, 128-141) was purified 15,000-fold to apparent homogeneity from human placenta. The soluble enzyme is a homodimer with a native molecular mass of 44-45 kDa. It has a pH optimum between 6.0 and 6.5 and is absolutely dependent on Mg2+ ions. The enzyme dephosphorylates certain 2'-, 3'-, and 5'-nucleotides. Km values for 2'- and 3'-nucleotides are around 0.3 mM with no preference for either ribo- or deoxyribonucleotides. 5'-Deoxyribonucleotides are 10-fold better substrates than the corresponding ribonucleotides, with dIMP greater than dUMP greater than dGMP greater than dTMP. dAMP is a poor substrate, dCMP is essentially insert. In all cases the Km values are in the millimolar range. Of the different forms of nucleotidases characterized in animal cells, the 5'(3')-nucleotidase is unique in its preference for 5'-deoxyribonucleotides. In intact cells, a portion of de novo synthesized deoxyribonucleotides is degraded as part of a homeostatic mechanism regulating the size of deoxyribonucleotide pools. This requires the participation of one or several 5'-nucleotidases. The 5'(3')-nucleotidase may be one such enzyme.