Isolated rat adipocytes were photo-affinity-labelled with B2-(4-azido-2-nitrophenylacetyl)-des-PheB1-insulin or B29-(4-azido-2-nitrophenylacetyl)insulin. Four anti-insulin antibodies (3 monoclonal, 1 polyclonal) were tested for their ability to inhibit the persistent stimulation of lipogenesis caused by the covalently bound insulin [Brandenburg et al. (1980) Nature (London) 286, 821-822]. The polyclonal and 2 monoclonal antibodies, directed against the C-terminus of the B-chain, gave a significant depression, while one antibody, directed against the region A(8-10), was without effect. Under reversible conditions, without irradiation, all antibodies completely inhibited lipogenesis. For the polyclonal antibody this is shown in a dose-dependent way. It is concluded that the effective antibodies can recognize their epitope because it is accessible on the surface of the complex and does not represent part of the receptor-binding surface of insulin. This binding leads to interference with the generation and/or transmittance of the biological signal.