The configuration of the substituents around the macrocycle core has been determined for acrylochlorin, the reduced porphyrin that is the iron-free form of heme d1. This has been accomplished by two independent techniques. Nuclear Overhauser enhancements on the proton resonances of acrylochlorin d3-methyl ester were measured to determine nearest-neighbor substituents and mesoprotons. A lanthanide shift reagent, tris-(1,1,1,2,2,3,3-heptafluoro-7,7-dimethyloctane-4,6-di onato) europium(III), was found to bind in the vicinity of the carbonyl oxygens of the propionate and acrylate methyl esters. Differential chemical shift effects on the proton resonances of acrylochlorin provided evidence on relative distances from the lanthanide-binding site. Since the substituents are constrained along the circumference of the macrocycle, this provided information on configuration. Assuming that the dominant mechanism for spin-lattice relaxation in acrylochlorin in deuterated chloroform solution is proton dipole-dipole interaction, measured values of spin-lattice relaxation times could be interpreted as being consistent with the proposed configuration. The configuration bears sufficient similarity to that of protoporphyrin IX so that a common nomenclature may be used (Smith K. M. (ed) (1975) Porphyrins and Metalloporphyrins, pp. 3-5, Elsevier/North-Holland Biomedical Press, Amsterdam). The deduced configuration of the acrylochlorin methyl ester is: 1-methyl, 1-hydroxymethyl, 2-methyl, 2-hydroxymethyl, 3-methyl formate, 4-methyl formate, 5-methyl, 6-methyl propionate, 7-methyl acrylate, 8-methyl. The naturally occurring heme d1 has free carboxylic acids at the 3-, 4-, 6-, and 7-positions.