We sought to determine whether the proliferation of immune cells affects the cellular uptake of the vitamin biotin. Peripheral blood mononuclear cells (PBMC) were isolated from healthy adults. The proliferation of PBMC was induced by either pokeweed lectin, concanavalin A, or phytohemagglutinin. When the medium contained a physiological concentration of [³H]biotin, nonproliferating PBMC accumulated 406 ± 201 amol [³H]biotin ⋅ 10⁶cells⁻¹ ⋅ 30 min⁻¹. For proliferating PBMC, [³H]biotin uptake increased to between 330 and 722% of nonproliferating values. Maximal transport rates of [³H]biotin in proliferating PBMC were also about four times greater than those in nonproliferating PBMC, suggesting that proliferation was associated with an increase in the number of biotin transporters on the PBMC membrane. The biotin affinities and specificities of the transporter for proliferating and nonproliferating PBMC were similar, providing evidence that the same transporter mediates biotin uptake in both states. [¹⁴C]urea uptake values for proliferating and nonproliferating PBMC were similar, suggesting that the increased [³H]biotin uptake was not caused by a global upregulation of transporters during proliferation. We conclude that PBMC proliferation increases the cellular accumulation of biotin.