The aim was to determine cadmium (Cd) speciation in various plants, between buffer-soluble and acid-soluble Cd, and also within the buffer-soluble Cd. A better understanding of Cd speciation shows the relative importance of different biological mechanisms for Cd sequestration. Roots of Pistia stratiodes, Eichhornia crassipes, Agrostis gigantea, Deschampsia caespitosa and wheat Triticum turgidum var. durum were analyzed. Buffer extractions solubilized varying proportions of Cd, ranging from 12% in Eichhornia to 83% in Agrostis. The proportion increased with time of Cd exposure in Pistia. It also increased in wheat roots as the external Cd rose from 0.05 to 0.5 muM and was lowest in old leaves and highest in roots. The remaining Cd was extractable with acid. Gel filtration resolved buffer-soluble Cd into three peaks distinct from inorganic Cd. Two complexes with phytochelatins and related polythiols were present in all cases, inorganic Cd being prominent only in Eichhornia extracts. The phytochelatin complexes accounted for 2% of the root Cd in Eichhornia to 78% in Agrostis. In wheat, phytochelatins bound 82% of the Cd in roots, 19% in young leaves and 12% in old leaves. The cysteine-rich protein metallothionein from wheat was detected immunologically in the void volume of gel filtrations of old and young leaves, but not of roots, and was distinct from the two phytochelatin-based complexes. Speciation of Cd in the various plants indicated that phytochelatins were not necessarily the major ligands of Cd.