Quiescent 3Y1 cells in monolayer cultures were dispersed with trypsin-EDTA, suspended in various media, and the cellular heat production was measured in a flow-type microcalorimeter set at 37°C. A linear relationship was found to exist between the number of cells applied to the microcalorimeter and the heat output. Increasing concentrations of bovine serum albumin (BSA) and of fetal calf serum (FCS) added in Dulbecco's modified Eagle's medium (DEM) enhanced the heat output to the same saturation level. Trypsin inhibitor added in DEM enhanced the heat output, but to a lower saturation level than FCS or BSA did, indicating that BSA has an activity to enhance cellular heat production by a mechanism other than neutralizing residual trypsin. The heat output was found to gradually decrease in the microcalorimeter. This reduction was not enhanced by a two-fold dilution of the medium (DEM plus FCS) with phosphate-buffered saline, indicating that this reduction is not caused by the depletion of nutrients and serum factors in the medium. Similarly, when cells were incubated for 155 or 220 min in suspension in DEM plus BSA at 37°C and applied to the microcalorimeter, the heat output decreased. However, no signifi-cant reduction of the heat output was observed after holding the cells at 0°C in suspension for the same period. This and other facts suggest that depletion of O₂ dissolved in the medium is involved in the gradual decrease in heat output. The present results will be especially useful in a future investigation in which we plan to measure the heat production in cells of a monolayer culture by flow microcalorimetry in an attempt to determine one of the parameters of the cellular physiological state.