The heat released from rat 3Y1 fibroblasts was measured by the stopped-flow method using a flow microcalorimeter. The heat output from a monolayer culture is defined as the maximum heat output from the cell suspension, after dispersion of the monolayer. The heat output per cell was not significantly changed when exponentially proliferating cells ceased dividing, with a G1 DNA content, at confluent cell density. When the growth-arrested (resting) cells were exposed to fresh medium containing various combinations of growth factors, heat output increased in parallel with the fraction of the cells that entered S phase of the cell cycle. The increase in heat output was correlated with the increase in cell volume. We infer from these findings that the resumption of cell proliferation stimulated by growth factors involves an increase in cell volume and a concomitant increase in heat production, but that entry into the resting state does not involve abrupt decreases in cell volume and heat production.