We have investigated the mechanisms whereby lipogenesis is markedly suppressed in adipose tissue depots of lactating sheep. Expression of the gene encoding acetyl-CoA carboxylase (ACC), the flux-determining enzyme of the lipogenic pathway, is reduced approximately threefold in both omental and subcutaneous adipose tissue depots during late pregnancy and remains so into lactation when compared with non-pregnant, non-lactating animals. By comparison, total ACC enzyme activity in these adipose depots is suppressed approximately 25- to 30-fold in lactation. Culture of explants from the subcutaneous depot of lactating sheep with insulin plus dexamethasone for 72 h resulted in an approximately sevenfold increase in ACC mRNA, a fivefold increase in total enzyme activity and a marked increase in the proportion of the enzyme in the active state when compared with explants cultured with no added hormones for the same period. However, there was a lag of between 32 and 48 h before marked induction of any of these parameters by insulin plus dexamethasone was observed. Induction of the alpha-tubulin gene paralleled that of the ACC gene, suggesting that cytoskeletal rearrangements are associated with the aquisition of sensitivity to insulin plus dexamethasone. These results demonstrate that the reduction in lipogenic capacity in ovine adipose tissue during lactation is related to repression of the ACC gene, both at the level of steady-state mRNA abundance and possibly at translation, as well as to suppression of the mechanisms that regulate the proportion of ACC in the active state, and these are further related to the marked insensitivity of these parameters to insulin plus dexamethasone in vitro.