Ouabain-like factor (OLF), a mammalian cardenolide, is a counterpart to plant-derived ouabain and is found in the adrenal, hypothalamus, and blood of several mammalian species. We now report the existence of a mammalian lactone-hydrogenated ouabain-like factor (dihydro-OLF) in secretions from cultured mouse adrenal Y-1 cells. Dihydro-OLF structurally and functionally mimics plant-derived dihydroouabain. We measured both OLF and the newly discovered dihydro-OLF using five independent techniques: immunoreactivity with two specific antisera, one against ouabain and one against dihydroouabain; chromatographic mobility; spectral absorbance characteristics; and concentration-dependent inhibition and phosphorylation of Na,K-adenosine triphosphatase. All measured physical attributes of dihydro-OLF mimic those of plant-derived dihydroouabain, including a spectral shift maxima, 220 nm (OLF) to 196 nm (dihydro-OLF), with appropriately decreased molar absorptivity. Dihydro-OLF (IC50 = 590 nM) is a 10-fold less potent Na+,K+-adenosine triphosphatase inhibitor than its oxidized mammalian counterpart OLF (IC50 = 60 nM), just as dihydroouabain is less potent than ouabain. Dihydro-OLF is also 3-fold more potent than a recently identified isomer of plant-derived dihydroouabain (IC50 = 1,700 nM). Using antiouabain and antidihydroouabain antisera we estimate that 3 x 10(7) mouse adrenal Y-1 cells secreted 1.3 ng OLF and 8.9 ng dihydro-OLF. The relative abundance of dihydro-OLF is consistently greater than that of its oxidized form, OLF, in bovine adrenals (22-fold), human serum (13-fold), and secretions from cultured mouse Y-1 cells (5-fold). The discoveries of OLF, OLF-genin, and now dihydro-OLF constitute an intriguing structural polymorphism probably involved in the synthesis, regulation, and metabolic control of these new hormone-like compounds.