Nucleoside analogs act as prodrugs that must be converted to 5'-phosphates by intracellular kinases to become active in the treatment of viral and oncological diseases. Activation may be reversed by dephosphorylation if the 5'-phosphates are substrates for 5'-nucleotidases. Dephosphorylation by cytosolic enzymes decreases the efficacy of the analogs, whereas dephosphorylation by mitochondrial enzymes may decrease mitochondrial toxicity. Both effects may influence the outcome of therapy. We investigated the dephosphorylation of the 5'-phosphates of commonly used nucleoside analogs by two cytosolic (cN-II and dNT-1) and one mitochondrial (dNT-2) nucleotidase. Most uracil/thymine nucleotide analogs were dephosphorylated by all three human enzymes but cytosine-containing nucleotide analogs were inactive. Only cN-II showed some activity with the monophosphates of the two purine analogs 2-chloro-2'-deoxyadenosine and 9-beta-D-arabinosylguanine. We conclude that overproduction of any of the three 5'-nucleotidases cannot explain development of resistance against cytosine analogs but that overproduction of cN-II could lead to resistance against purine analogs. Of the tested analogs, only (E)-5-(2-bromovinyl)-2'-deoxyuridine was preferentially dephosphorylated by mitochondrial dNT-2. We propose that in future developments of analogs this aspect be considered in order to reduce mitochondrial toxicity. We tested inhibition of dNT-1 and dNT-2 by a large variety of synthetic metabolically stable nucleoside phosphonate analogs and found one (PMcP-U) that inhibited dNT-1 and dNT-2 competitively and a second (DPB-T) that inhibited dNT-2 by mixed inhibition. Both inhibitors are useful for specific 5'-nucleotidase assays and structural studies and may open up possibilities for therapy.