Stimulatory effect of serotonin on lactate production of rat diaphragm was examined in Krebs-Ringer bicarbonate solution. At a dose of 50 ug per flask, clear-cut effect obtained when incubated with diaphragm from glucose-fed rat, but inconsistent result was noted in diaphragm from fasted rats. When dose of serotonin was raised to 200 - 250 ug, lactate production was stimulated even in fasted rat diaphragm. Simultaneous occurrence of enhanced glucose uptake and glycogenolysis was observed when 200-250 ug serotonin was added, whereas increase in lactate production elicited with 50 ug serotonin was never accompanied by an increase of either glucose uptake or glycogenolysis. Results suggest that serotonin, in contrast to insulin or epinephrine, more or less directly increases lactate output from endogenous intermediaries.