Progesterone and some derivatives were tested in a radioimmunoassay (RIA) of digoxin and in a bioassay measuring the 86Rb-uptake into red blood cells as an index of Na+, K+-ATPase activity. The digitalis-like activity of the hormones was compared with that found in chromatographic fractions of material extracted from the urines of pregnant women at term. Progesterone at concentrations greater than 10(-6) M cross-reacted in the RIA, and at 10(-3) M it decreased 86Rb-uptake by 18%. The anaesthetic progesterone derivates 5 alpha-pregnane-3 alpha-ol-20-one and 5 alpha-pregnane-3,20-dione crossreacted to a lesser degree in the RIA and lacked effect in the bioassay. Similar results were obtained with pregnandiol-glucuronide, the major urinary metabolite of progesterone. In contrast, several fractions of the urinary material had significant effects in both assays. It is concluded that the digitalis-like activity of progesterone is not coupled to properties associated with its anaesthetic effects. Furthermore, although progesterone may account for a part of the endogenous digoxin-like substances in serum of neonates and pregnant women, neither progesterone proper nor pregnandiol-glucuronide explains the great amount of digoxin-like substances found in the urines.