Two isoenzymes of p-coumarate: CoA ligase were isolated from cell suspension cultures of soybean (Glycine max L., var. Mandarin). Separation and partial purification of the enzymes were achieved by precipitation with MnCl2 and (NH4)2SO4, and column chromatography on DEAE-cellulose, Sephadex G-100 and hydroxyapatite. The isoenzymes had approximately the same molecular weight, but differed significantly with respect to their substrate specificity, their inhibition constants for AMP, their dependence on pH and ionic strength for optimum activity, and their fractionation pattern during the purification procedure or upon analytical disc-gel electrophoresis. Both coumarate: CoA ligases were specific for the activation of various substituted cinnamic acids. Of the cinnamic acids tested, ferulic, sinapic, 5-hydroxyferulic, p-coumaric, and caffeic acids were the substrates with the lowest apparent Km values (on all the order of 1 to 4 x 10(-5) M) for isoenzyme 1. The lowest apparent Km values (from about 1 to 9 x 10(-5) M) for isoenzyme 2 were obtained for caffeic, p-coumaric, m-coumaric, and o-coumaric acids. Sinapic acid and several methoxycinnamic acids were efficient substrates of isoenzyme 1 but were not activated at all by isoenzyme 2. The possible roles of the two p-coumarate: CoA ligase isoenzymes in the phenylpropanoid metabolism of the cell cultures are discussed.