Two splice variants of the human SLC30A5 Zn transporter gene include different first exons, indicating transcription from alternative promoters. Splice variant A is expressed at the apical membrane of transiently transfected Caco-2 cells (Cragg et al., 2002), whereas the intracellular location of splice variant B is unknown. We previously reported that the 2.9 kb genomic region immediately upstream of the first exon of splice variant A failed to drive Zn-inducible expression of a reporter gene in the placental cell line JAR, consistent with our observations that SLC30A5 mRNA in JAR cells was not altered by changing the extracellular Zn concentration (Russi & Ford, 2003). We now present evidence that this genomic region drives Zn-regulated expression of a reporter gene in the human intestinal cell line Caco-2, consistent with earlier observations that increasing the extracellular Zn concentration increased SLC30A5 mRNA in Caco-2 cells (Cragg et al., 2002). We also demonstrate that, whilst neither splice variant of SLC30A5 is regulated by Zn in JAR cells, splice variant A is up-regulated and splice variant B is down-regulated in Caco-2 cells cultured at an increased Zn concentration. A 2024 bp genomic region immediately upstream of the first exon of SLC30A5 splice variant A, including the 5′ end of the cDNA, was subcloned into pBlue TOPO upstream of the E. coli β-galacotosidase reporter gene to give the plasmid pC2024. pC2024 was transfected into Caco-2 cells for transient expression. The Zn concentration of the culture medium was modified 24 hours post-transfection and β-galactosidase activity was measured in cell lysates after a further 24 hours. β-galactosidase activity expressed from pC2024 in Caco-2 cells at 100 μM Zn was greater than that expressed at 3 μM Zn (5.2 ± 0.8 compared with 2.4 ± 0.5, mean ± SEM in units of fluorescence/mg protein; n=12, P< 0.05 by Student's t test). Analysis of RNA by northern blotting followed by denistometric analysis of band intensities revealed that in Caco-2 cells SLC30A5 splice variant A was up-regulated 2.0-fold but splice variant B was down-regulated 2.2-fold at 100 μM compared with 3 μM Zn. Western blotting revealed a parallel differential regulatory effect of two different molecular weight species at the protein level. In JAR cells neither splice variant was regulated by the change in Zn concentration. The expression of differentially regulated SLC30A5 splice variants, possibly mediated through the use of alternative promoters, may contribute to cellular and/or systemic Zn homeostasis.