This brief overview describes some of the properties of various cellular phosphotransferase systems, with particular emphasis on nucleoside 5'-triphosphate (NTP)-dependent protein kinases and nucleoside kinases, for which it is widely and implicitly assumed that ATP is the intracellular phosphate donor. Numerous examples are presented, based on the in vitro properties of these enzymes, to show that ATP is not the only, or frequently not even the major, phosphate donor, and that this is probably reflected in vivo. It is pointed out that in vitro studies of donor and acceptor specificities of kinases must take account of the intracellular concentrations of nucleoside 5'-triphosphates, a problem also relevant to the design of nucleoside analogues as chemotherapeutic agents. Attention is also drawn to NTP analogues as substrates/ inhibitors of protein kinases, and to several examples of low-molecular mass non-peptide substrates of these enzymes.