Zinc is a vital micronutrient to all organisms, but is also a toxicant to aquatic species. It is therefore of importance to determine the mechanisms by which zinc uptake is modulated. In the present study, we investigated the regulatory effects of the vitamin D metabolite, 1alpha,25-(OH)(2)D(3), on branchial zinc influx in rainbow trout, Oncorhynchus mykiss. Our results showed that branchial zinc uptake in rainbow trout was stimulated 7 days after a single intraperitoneal injection of 1alpha,25-(OH)(2)D(3) (0.01 microg/g fish). To understand the molecular components of zinc uptake regulation by 1alpha,25-(OH)(2)D(3), a ZIP zinc transporter (OmSLC39A1) and a partial vitamin D receptor (OmVDR) were molecularly cloned from rainbow trout gill, and the transcriptional expression of OmSLC39A1, epithelial calcium channel (OmECaC) and OmVDR genes in the gill was subsequently analyzed in response to 1alpha,25-(OH)(2)D(3). OmECaC, OmSLC39A1 and OmVDR were all upregulated following treatment with 1alpha,25-(OH)(2)D(3), but the effect was observed at different time points. OmECaC expression was significantly increased by 1alpha,25-(OH)(2)D(3) on Days 3 and 5 after the injection, and expression of OmVDR was stimulated on Day 5. There was also an increased abundance of OmSLC39A1 mRNA on Day 7 following the injection with 1alpha,25-(OH)(2)D(3), but given the late response the effect of 1alpha,25-(OH)(2)D(3) on this gene might be indirect. The results from the present study provide strong evidence that administration of 1alpha,25-(OH)(2)D(3) results in enhanced zinc uptake across rainbow trout gill and that this effect is associated with an increased expression of transporters that mediate zinc uptake. The implications of our findings, in terms of aquatic toxicology, are that vitamin D status influences zinc accumulation in gill and body of fish.