The biologic and chemical properties of three photoreactive insulin derivatives, NαA1-(2-nitro-4-azido-phenyl) insulin (A1-Nap-Ins), NαB1-(2-nitro-4-azidophenyl acetyl) insulin (B1-Napa-Ins), and NεB29-(2-nitro-4-azidophenyl acetyl) insulin (B29-Napa-Ins), were characterized in isolated rat adipocytes. In the absence of photolysis, the relative affinities of these three analogues for the insulin receptor were 0.07, 1.0, and 0.55 when compared with native beef insulin, and these correlated well with the ability of these analogues to stimulate glucose oxidation in a fat cell bioassay. After photolysis and extensive washing to remove noncovalently bound insulin, the Bland B29-photoreactive insulins produced a persistent biologic effect in the isolated adipocytes, with up to 90% of the maximal stimulatory level of insulin. By contrast, photolysis did not produce a persistent biologic effect of the A1-Nap-Ins, even when its concentration was increased 10-fold to account for the difference in receptor affinity. All three analogues were iodinated and used, for affinity labeling of the insulin receptor in isolated adipocytes. The structure of the insulin receptor was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis and autoradiography. When gels were run under reducing conditions, all three analogues labeled predominantly a protein with an apparent mol wt (Mr) of 138,000, corresponding to the a subunit of the insulin receptor. Minor components with apparent Mr = 90-120K were also identified using both the B1 and B29 analogues, and a minor band was observed at 45K with the B29-Napa-Ins only. The A1-Nap-Ins also strongly labeled a component of Mr = 85,000; this was, however, only partially inhibited by insulin. Under honreducing conditions, the photoinsulins were linked to receptor oligomers of Mr = 330-350K, 220K, and 185K, as well as to a small amount of free a subunits. With increasing reductant concentrations, these oligomers decreased and the intensity of the a subunit band increased. These data suggest that although all three photoinsulins bind predominantly to the a subunit, only those linked via the B-chain produce a persistent biologic effect. These studies also suggest that the insulin receptor of isolated fat cells in the native state exists in various oligomeric forms, as well as free subunits, in the plasma membrane.