Intracellular zinc signaling is important in the control of a number of cellular processes. Hormonal factors that regulate cellular zinc influx and initiate zinc signals are poorly understood. The present study investigates the possibility for cross talk between the glucocorticoid and zinc signaling pathways in cultured rainbow trout gill epithelial cells. The rainbow trout metallothionein A (MTA) gene possesses a putative glucocorticoid response element and multiple metal response elements 1042 base pairs upstream of the start codon, whereas metallothionein B (MTB) and zinc transporter-1 (ZnT1) have multiple metal response elements but no glucocorticoid response elements in this region. Cortisol increased MTA, MTB, and ZnT1 gene expression, and this stimulation was enhanced if cells were treated with cortisol together with zinc. Cells treated with zinc showed increased zinc accumulation, transepithelial zinc influx (apical to basolateral), and intracellular labile zinc concentrations. These responses were also significantly enhanced in cells pretreated with cortisol and zinc. The cortisol-mediated effects were blocked by the glucocorticoid receptor (GR) antagonist RU-486, indicating mediation via a GR. In reporter gene assays, zinc stimulated MTA promoter activity, whereas cortisol did not. Furthermore, cortisol significantly reduced zinc-stimulated MTA promoter activity in cells expressing exogenous rainbow trout GR. These results demonstrate that cortisol enhances cellular zinc uptake, which in turn stimulates expression of MTA, MTB, and ZnT1 genes.