The phorbol nucleus was succinylated and then conjugated to bovine albumin using dicyclohexylcarbodiimide. Rabbits given injections of the conjugate developed antibodies which rose in titer progressively with repeated immunization. By the ninth bleeding, the binding of one antiserum, diluted 1:15,000, was saturated with about 10 nM [3H]phorbol-12,13-dibutyrate ([3H]PDBU) and had an average association constant, Ka, of 2.6 x 108 M~V The serological specificity of the antisera was charac terized by examining the inhibition of the [3H]PDBU-anti-phorbol Buccinate immune system by 18 phorbol-related compounds. The specificities of antibodies from two rabbits tested in detail were qualitatively similar. The rank order of inhibitory activity for certain phorbol-related compounds was PDBU [concentration of inhibitor required to give 50% inhibition of PDBU binding (ICso)= 7.6 nM] = phorbol-13-acetate [ICso = 8.2 nw] > phorbol-12,13dibenzoate > 4-/3-phorbol [ICso = 124 nM] > phorbol-12,13diacetate > phorbol-12-myristate-13-acetate [ICso = 184 nM] > phorbol-13,20-diacetate > phorbol-12-acetate [ICso = 2300 nM]. The following compounds showed no detectable serological activity: mezerein, 4-O-methylphorbol-12-myristate-13-acetate, ingenol, 4-a-phorbol, teleocidin B, and dihydroteleocidin B. These and other results indicated that the 4-/3-phorbol nucleus was required for serological activity, that esterification of the C-13 position with benzoate, acetate, or butyrate enhanced the immunoreactivity of 4-/3-phorbd, and that among the phorbolrelated compounds examined there was no direct relationship between serological activity and biological potency as tumor promoters. Using the [3H]PDBU-anti-phorbol succinate immune system, we measured the concentrations of immunoreactive phorbol-related material in crude mixtures such as croton oil and performed pharmacokinetic studies in rats given PDBU s.c.