Characterization of non-transferrin (non-Tf) iron transport by K562 cells has revealed unique properties relative to iron uptake mechanisms present in other cell types (Inman, R. S., and Wessling-Resnick, M. (1993) J. Biol. Chem. 268, 8521-8528). Since treatment of K562 cells with phorbol esters promotes stable megakaryocytic differentiation, we examined the uptake of non-Tf iron in response to protein kinase C activation. Treatment of K562 cells with phorbol esters increased the cellular uptake of 55Fe 4-6-fold compared with untreated cells. The phorbol ester-induced stimulation of 55Fe uptake was time- and dose-dependent, with significantly enhanced transport observed only after prolonged administration of 50 nM phorbol 12,13-dibutyrate (> 8 h). These effects can be attributed to an increased Vmax of transport (14.0 +/- 5 versus 0.6 +/- 0.2 pmol/min/10(6) cells) as well as a 6-fold increase in the apparent Km (1.2 +/- 0.4 versus 0.2 +/- 0.06 microM). It is thought that the reduction of Fe3+ to Fe2+ is required as a first step in the uptake mechanism, and the associated ferrireductase activity of K562 cells is also enhanced with phorbol ester treatment by 5-10-fold (337 +/- 53 versus 43 +/- 3 pmol/min/10(6) cells). Bryostatin-1, a protein kinase C activator that fails to induce differentiation of K562 cells, did not promote this effect, indicating that the enhanced transport activity is dependent on the differentiation response. The idea that synthesis of a new class of transporters is responsible for this effect is supported by the observation that actinomycin D blocks up-regulation of non-Tf iron transport. The increased transport and ferrireductase activity induced upon differentiation also correlate with the appearance of saturable iron-binding sites on the surface of K562 cells with Kd approximately 0.4 microM. These results indicate that non-Tf iron transport activity and the expression of cell-surface iron-binding proteins can be controlled by environmental factors that promote megakaryocytic differentiation of K562 cells.