Time-related alterations in a digitalis-like factor in urine were examined by means of cross-reactivity with an anti-digoxin antibody during the development of hypertension in DOCA-salt rats. Daily urinary sodium excretion was also measured. After hypertension had developed, plasma levels of the digitalis-like factor were determined by two methods: radioimmunoassay for digoxin and a receptor binding assay using 3H-ouabain and a rat brain synaptosomal protein. Urinary digoxin-like immunoreactivity increased gradually and significantly in the DOCA-salt rats as compared with that of sham-operated high-salt rats and normal-salt rats. Urinary sodium excretion was significantly higher in the DOCA-salt rats, and a significant correlation (r = 0.56, p less than 0.001) was observed between the daily urinary digoxin-like immunoreactivity and daily sodium excretion. In plasma, both digoxin-like immunoreactivity and ouabain-like binding activity were significantly higher in the DOCA-salt rats than in the other 2 groups. These results suggest that digitalis-like factor plays an important role in the development of hypertension in DOCA-salt rats.