Tomato is a model plant for studying molecular mechanisms of iron uptake and metabolism in strategy I plants (dicots and non-graminaceous monocots). Reduction of ferric to ferrous iron on the root surface is an obligatory process for iron acquisition from soil in these plants. LeFRO1 encoding an Fe(III)-chelate reductase protein was isolated from the tomato genome. We show that expression of LeFRO1 in yeast increases Fe(III)-chelate reductase activity. In a transient expression analysis we found that LeFRO1 protein was targeted on the plasma membrane. LeFRO1 transcript was detected in roots, leaves, cotyledons, flowers and young fruits by RT-PCR analysis. Abundance of LeFRO1 mRNA was much lower in young fruits than in other tissues. The transcription intensity of LeFRO1 in roots is dependent on the iron status whereas it is constitutively expressed in leaves. These results indicate that LeFRO1 is required in roots and shoots as well as in reproductive organs for iron homeostasis and that its transcription in roots and shoots is regulated by different control mechanisms. The expression of LeFRO1 was disrupted in the iron-inefficient mutants chloronerva and T3238 fer, indicating that FER and CHLN genes are involved in the regulation of LeFRO1 expression in tomato roots. The differential expression of LeFRO1 and LeIRT1 (an iron-regulated metal transporter gene in tomato) in roots of T3238 fer under iron-deficient and -sufficient conditions suggests that the FER gene may regulate expression of LeFRO1 more directly than that of LeIRT1 in tomato roots.