A method that employs gas chromatography-mass spectrometry has been developed to measure N tau-methylhistidine (3-methylhistidine; 3-MH) synthesis and release from skeletal muscle myotubes in vitro. It shows excellent linearity (0.9999) over the range studied (0-4 nmol), high recovery (92.6%), and low coefficient of variation (1.6%). 3-MH release from myotubes was essentially linear over a 96-h incubation, whereas the loss of 3-MH from cell protein accelerated with increasing time, an effect due, at lest in part, to decreasing rates of total protein synthesis. When incubated in either glutamine-free or methionine-free medium for 48 h, 3-MH in cell protein and appearing in the medium were greatly reduced compared with the 48-h controls, suggesting that hypertrophy was greatly reduced. Similar but lesser trends were observed with adenosine 3',5' -cyclic monophosphate. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) appeared to both stimulate 3-MII synthesis and inhibit its release during a 48-h incubation. The development of this method facilitates detailed investigation into the mechanisms through which agents such as TPA regulate myofibrillar protein degradation.