Earlier studies have shown that transferrin-gallium inhibits cellular iron incorporation and blocks DNA synthesis by decreasing the activity of the iron-dependent M2 subunit of ribonucleotide reductase. We examined the growth-inhibitory effects of gallium nitrate in combination with clinically relevant inhibitors of ribonucleotide reductase fludarabine (an M1 subunit inhibitor), and iron chelators (M2 subunit inhibitors). Fludarabine and gallium nitrate in combination produced a significant increase in cell growth inhibition when compared with either agent alone; however, this effect was partially reversible up to 24 h and was best seen with continuous exposure of cells to both drugs. Incubation of cells with desferrioxamine and gallium nitrate resulted in reversal of gallium-induced growth inhibition. Incubation of cells with N,N'-bis(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid and gallium nitrate resulted in a slight increase in gallium-induced growth inhibition, with partial restoration of cell growth occurring only at a single high concentration of N,N'-bis(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid. Both chelators inhibited 67Ga uptake by cells and increased cell surface transferrin receptors. In contrast to the coincubation studies, sequential exposure of cells to desferrioxamine or N,N'-bis(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid and gallium nitrate resulted in a significant potentiation of the growth-inhibitory effects of gallium nitrate. Our studies show that cellular iron deprivation results in enhanced sensitivity of cells to gallium. Furthermore, the combined effects of fludarabine and gallium on cell growth may be of clinical relevance, since both agents are active against lymphoid cancers.