To estimate the turnover of UDP-N-acetylglucosaminyl transferase (OGT), we exposed stably transfected HeLa cells to tetracycline for 16h to induce OGT gene expression and increase cytosolic enzyme levels. Removal of tetracycline led to a progressive decrease in OGT activity (after a 6h lag period), yielding an estimated OGT half-life of 13h. A similar half-life (12h) was obtained by measuring the loss of biosynthetically labeled OGT ([35S]methionine pulse-chase experiments). Since OGT turnover was relatively slow, it is unlikely that changes in OGT gene expression or protein expression play a role in the short-term regulatory actions mediated by the hexosamine signaling pathway. We also found that the overexpressed 110kDa murine OGT subunit (recombinant enzyme) was enzymatically similar to the endogenous holoenzyme derived from rat brain tissue. Thus, stably transfected HeLa cells provide an abundant source of enzyme that can be used to study the structure, function, and regulation of OGT.