Iron is essential for male fertility and disruptions in iron balance lead to impairment of testicular function. The divalent metal transporter DMT1 is a key modulator of transferrin and non-transferrin bound iron homeostasis. As a first step in determining the role of DMT1 in the testis, we have determined the pattern of DMT1 expression in the developing and adult rat testis. Northern analysis and RT-PCR of testis polyadenylated RNA revealed the presence of IRE and non-IRE transcripts. Semi-quantitative immunoblotting of immature and adult rat testis uncovered the expression of two distinct DMT1 protein species. Immunohistochemistry showed DMT1 was widespread throughout each seminiferous tubule and was expressed in the intracellular compartment. In the adult rat testis DMT1 was immunolocalised to both the Sertoli and the germ cells. In contrast to the immature testis, expression was dependent on the stage of the spermatogenic cycle. DMT1 was not detected on any plasma membranes in either the developing or adult testis, suggesting that DMT1 is not primarily responsible for translocating iron across this epithelia. Our data suggests an important role for DMT1 in intracellular iron handling during spermatogenesis and implies that germ cells have a need for a precisely targeted and timed supply of iron. We suggest that DMT1 may, like in other tissues, play a role in transporting iron between intracellular compartments and thus play an important role in male fertility.