The aim of the study was to further investigate the nature of endogenous digitalis-like factors stimulated by acute plasma volume expansion (VE). 10 male Fisher 344XNB rats were given intravenous (iv) saline infusion (4% of the body mass) for 30 min, which caused a fall in % hematocrit (35.9+/-0.69 vs. 41.8+/-1.05; vs. 10 controls, P<0.01). EDLF was measured in C-18 reverse-phase extracted (32%+80% acetonitrile) tissues. VE was associated with an increase in plasma marinobufagenin-like factor (MLF)(0.49+/-0.05 vs. 0.2+/-0.06 nmol/L, P<0.01) and pituitary ouabain-like compound (OLC) (30.9+/-3.12 vs. 3.2+/-2.3 pmol/g, P<0.01). Plasma OLC decreased (0.087+/-0.018 vs. 0.21+/-0.04 nmol/L, P<0.02), and pituitary MLF did not change (0.05+/-0.01 vs. 0.07+/-0.02 pmol/g) after VE. Chloroform extracted urine from 5 volume-expanded male mongrel dogs was fractionated on reverse-phase HPLC columns in a linear gradient of 0-80% acetonitrile. The material cross-reacting with marinobufagenin antibody eluted from HPLC column as a single peak, demonstrated absorbance at 300 nm, and was distinct from ouabain-like material. Digoxin antibodies interacted with both marinobufagenin-like and ouabain-like immunoreactive material. These results provide further evidence for the presence of a bufodienolide EDLF in the mammals, and demonstrate that EDLF response to VE includes stimulation of brain OLC and plasma bufodienolide.