We studied the uptake of biotin into human peripheral blood mononuclear cells (PBMC) using [³H]biotin and studied the catabolism of biotin in PBMC using [¹⁴C]biotin. Over 30 min, [³H]biotin uptake was greater at 37°C than at 25°C (K _T = 2.6 ± 0.4 nM, maximal velocity = 2.9 ± 0.2 fmol ⋅ 10⁶cells⁻¹ ⋅ 30 min⁻¹). Ouabain reduced [³H]biotin uptake to 65% of control values, suggesting that biotin uptake is Na-K-ATPase dependent. Unlabeled biotin and biotin analogs reduced the uptake of [³H]biotin to 22–70% of control values, suggesting the presence of a competition for a structurally specific biotin transporter. When endocytosis by PBMC was stimulated by various acyl glycerols, [³H]biotin uptake was 40–73% of control values; these data are consistent with the hypothesis that stimulated endocytosis reduces biotin transporter density on the cell surface. During a 168-h incubation, PBMC did not catabolize [¹⁴C]biotin.