Previous investigations suggested that binding of prostaglandin to a myoblast membrane receptor initiates a second messenger cascade which is essential for subsequent myogenesis. Initial evidence of the sensitivity of myogenesis to lithium suggested the involvement of inositol phosphate metabolism. That possibility is investigated here. The accumulation of inositol monophosphate in response to prostaglandin binding was studied in aggregate cultures of chick embryo myoblasts in vitro. At 22 or 28 h in culture mononucleated myoblasts were labeled with [3H]inositol, which was then incorporated into phosphoinositides. After experimental manipulations of prostaglandin metabolism and the addition of Li+ prior to prostaglandin binding at 33 h, [3H]inositol monophosphate accumulation was measured by anion-exchange chromatography between 33 and 37 h. Inositol monophosphate was found to accumulate rapidly following 33 h. However, after 36 h of myogenesis, no inositol monophosphate accumulation was observed. The accumulation was dependent on prostaglandin as indomethacin, which also blocks subsequent membrane events in myogenesis, blocked inositol phosphate accumulation. Like subsequent myogenesis, inositol phosphate accumulation was restored by the addition of exogenous prostaglandin. Finally, the accumulation of inositol phosphate began only after the binding of prostaglandin. The results demonstrate that an inositol phosphate signal transduction mechanism connects prostaglandin binding to membrane events in embryonic chick myogenesis.