ABSTRACT Zinc is an essential micronutrient that cells must obtain from the environment in order to develop their normal growth. Previous work performed at our laboratory showed that the synthesis of immunodominant antigens from Aspergillus spp., including A. fumigatus , was up-regulated by a low environmental concentration of zinc. These results suggested that a tightly regulated system for the fungus to grow under zinc-limiting conditions must underlie the ability of A. fumigatus to acquire zinc in such environments. In this work, we show that zrfA and zrfB are two of the genes that encode membrane zinc transporters from A. fumigatus in this system. Expression of these genes is differentially down-regulated by increasing concentrations of zinc in the medium. Thus, the transcription of zrfB is turned off at a concentration 50-fold higher than that for zrfA transcription. In addition, phenotypic analyses of single zrfA Δ and zrfB Δ mutants and a double zrfAzrfB Δ mutant revealed that the deletion of zrfB causes a greater defect in growth than the single deletion of zrfA . Deletion of both genes has a dramatic effect on growth under acid, zinc-limiting conditions. Interestingly, in neutral or slightly alkaline zinc-depleted medium, the transcriptional expression of both genes is down-regulated to such an extent that even in the absence of a supplement of zinc, the expression of zrfA and zrfB is strongly reduced. This fact correlates with the growth observed in alkaline medium, in which even a zrfAzrfB Δ double mutant was able to grow in a similar way to the wild-type under extremely zinc-limiting conditions. In sum, the zinc transport proteins encoded by zrfA and zrfB are members of a zinc uptake system of A. fumigatus that operates mainly under acid, zinc-limiting conditions.