The mechanisms of brown adipocyte proliferation and differentiation during cold acclimation (and/or adaptation to hyperphagia) have been studied by quantitative photonic radioautography. [3H]thymidine was injected to warm-acclimated (25 degrees C) rats and to animals exposed to 5 degrees C for 2 days. Samples of interscapular brown adipose tissue were collected for quantitative analysis of mitotic frequencies at various periods of time (4 h-15 days) after the injection of [3H]thymidine, the rats being maintained at the temperatures to which they were initially exposed. Confirming our previous results [Bukowiecki et al., Am. J. Physiol. 242 (Endocrinol. Metab. 5): E353-E359, 1982], it was found that cold exposure for 2 days markedly enhanced mitotic activity in endothelial cells, interstitial cells, and brown preadipocytes rather than in fully differentiated brown adipocytes. The total tissue labeling index (percent of labeled nuclei) increased approximately 70 times over control values. We now report that cellular labeling progressively increased in mature brown adipocytes during cold acclimation, whereas it correspondingly decreased in interstitial cells and brown preadipocytes. This indicates that the sequence of events for cellular differentiation is interstitial cells----brown preadipocytes----mature brown adipocytes. Remarkable, labeling frequency did not change in endothelial cells during cold acclimation demonstrating that these cells cannot be considered as progenitors of brown adipocytes. It is suggested that brown adipocyte proliferation and differentiation from interstitial cells represent the fundamental phenomena explaining the enhanced capacity of cold-acclimated and/or hyperphagic rats to respond calorigenically to catecholamines.