Zinc is an essential but potentially toxic nutrient. Little is known about how eukaryotic cells maintain viability in the face of up to 100,000-fold variations in environmental zinc. To help identify the mechanisms of zinc homeostasis in yeast, diploid cells were transformed with a genomic library containing random miniTn3-mediated lacZ insertions (N. Burns et al., Genes Dev. (1994) 8:1087) with the aim of isolating cells with zinc-regulated lacZ expression. Of approximately 80,000 independent transformants screened by replica-plating onto low (10 nM) and high (10 µM) zinc media, 57 clones expressed lacZ primarily on low zinc medium, while 52 other clones expressed lacZ primarily on high zinc medium. Characterization of the lacZ insertions in these clones was accomplished using an inverse PCR technique. Genes putatively induced by zinc deprivation included one gene encoding a zinc-finger regulatory protein; three genes encoding transport proteins, including ZRC1 and PMR2/ENA2; ten other known genes, including PHO13, MET30, and ADH4; and 14 open reading frames (ORFs). Genes regulated in the opposite direction included six known genes, including TSA, OYE3, and ADH3; six ORFs; and the ORFs in the retrotransposons Ty1 (31 clones). These results demonstrate the power of genome-wide methodologies for identifying differentially regulated genes in yeast and should lead to many new insights into the mechanisms of zinc homeostasis in eukaryotic cells.