In patients with chronic uremia we have previously demonstrated a significant inhibition of the Na-K-ATPase enzyme which represents the specific receptor protein for cardiac glycosides. Since an endogenous inhibitor of this enzyme was previously shown to react with a digoxin antibody, in the present study we determined digoxin-like immunoreacting activity(ies) (DLIA) by a radioimmunoassay in 15 nondialyzed patients with chronic renal failure. In native serum, DLIA ranged from 0 to 1.70 ng/ml and was unrelated to the degree of renal failure. After gel filtration of serum, DLIA exclusively eluted in the small molecular weight salt (F III) and post-salt (FIV) fractions and averaged 0.22 ± 0.04 and 0.20 ± 0.05 ng/ml in fractions III and IV, respectively. Total activities ranged from 0.11 to 0.88 ng/ml with a mean of 0.42 ± 0.06 ng/ml and closely correlated with the degree of renal impairment (p < 0.001). The results confirm the presence of small molecular weight digoxin-like immunoreacting substance(s) in uremic serum. The variable activities in native serum and the lack of correlation between the degree of renal failure and DLIA in serum fraction IV previously shown to possess the Na-K-ATPase-inhibiting activity, however, indicate that DLIA may not reflect specifically the endogenous sodium pump inhibitor and that unspecific binding to this digoxin antibody of uremic toxins or other endogenous compounds, such as steroids other than aldosterone, may have occurred.