It is widely but not unanimously accepted that one or more endogenous digitalis-like factors (EDLF) circulate in human plasma. In this paper we provide confirmatory evidence that newborn plasma contains a factor with immunological and biological properties similar to ouabain and demonstrate that this factor may be present also in the adult. In fact, we obtained in newborn and adult plasma extracts, identical HPLC elution profiles of ouabain-like immunoreactivity and 86Rb erythrocyte uptake inhibitory activity with a major peak corresponding to the retention time of ouabain. The fact that immunoreactivity and biological digitalis-like activity in the peak are due to an identical substance is strongly supported by the correlation between RIA and 86Rb uptake inhibitory values observed in the purified fractions. Finally, the strong correlation between immunoreactivity observed in plasma samples after simple SepPak C18 extraction and after additional HPLC suggests that less purified samples may be assayed for screening purposes. However, for a more quantitative assessment, this simple extraction method needs a subsequent HPLC purification for eliminating an overestimation of values due to cross-reacting impurities.