Physiological concentrations of retinoic acid can block the activation of human erythrocyte Ca(2+)-ATPase in vitro by thyroid hormone [Smith, Davis & Davis (1989) J. Biol. Chem. 264, 687-689]. The present studies were undertaken to ascertain the nature of this blockade. Two binding sites for L-thyroxine (T4) were demonstrated on washed erythrocyte membranes; the high-affinity site had a Kd value of 2.7 x 10(-10)M and a Bmax. of 76 fmol/mg of protein. The lower-affinity site possessed a Kd of 1 x 10(-8) M. Retinoic acid was as potent a displacer of radiolabelled T4 as was the unlabelled hormone. Certain retinoic acid analogues with either ring or fatty acid side chain modifications retained some ability to displace [125I]T4 binding and to block iodothyronine activation of Ca(2+)-ATPase. The side chain terminal carboxyl group was essential for full activity of the retinoic acid molecule. Its absence or replacement with an ethylsulphone group rendered the molecule considerably less active in the ATPase model. Retinol, 13-cis-retinoic acid, benzene-substituted all-trans-retinoic acid and polyprenoic acid all failed to influence iodothyronine binding or to block activation of Ca(2+)-ATPase by T4. There was good agreement between the ability of an analogue to displace [125I]iodothyronine binding and its ability to inhibit the T4-dependent activation of the Ca(2+)-ATPase. It would appear from these observations that retinoic acid can modulate the activation of erythrocyte membrane Ca(2+)-ATPase by thyroid hormone through a mechanism which involves displacement of iodothyronine from binding sites. These activities apparently derive from both the ring structure and the fatty acid side chain of the retinoic acid molecule.