In order to examine whether the differentiation process in brown adipocytes cultivated in primary culture is associated with substantial alterations in the complement of G proteins, the levels of these proteins were investigated with immuno-electrophoretic techniques in membrane preparations from proliferating and differentiated cultured mouse brown adipocytes. We observed that differentiation was associated with a dramatic (more than threefold) increase in the short variant of G s α protein (G s αS). The long variant of G s α (G s αL), as well as G i 1α, G i 2α, G q α, G 1 1 α and Gβ subunit proteins remained unchanged whereas G i 3α protein was decreased. These changes were accompanied by marked increase in isoprenaline-, forskolin- as well as manganese-stimulated adenylyl cyclase. Thus, the marked increase in β-adrenergic responsiveness of fully differentiated confluent brown adipocytes (day 8-9), as compared with that of proliferating undifferentiated cells of 'fibroblast phenotype' (day 3-4), is associated with a significant increase in the relative proportion between the short and long variants of G s α (the G s αS/G s αL ratio) along with a decrease in G i 3α protein. These data also suggest that the short variant of G s α exhibits higher functional activity than the long variant of this G protein.