An earlier study has shown that RNAi knock-down of a single organic anion transporter (OAT) gene in the principal cells of Drosophila Malpighian tubules is associated with reductions in the expression of multiple, functionally related genes. In this study, we measured the rates of secretion of four fluorescent ions by tubules isolated from flies expressing targeted RNAi knock-down of specific OAT genes. Droplets secreted by isolated tubules set up in the Ramsay assay were collected in optically flat capillary tubes and the concentrations of fluorescent ions were determined by confocal laser scanning microscopy. Reductions in the expression of organic anion (OA) transporting polypeptide 58Dc (OATP; CG3380) were associated with reduced secretion of the OAs fluorescein and Texas Red. Reduction in the expression of Drosophila multidrug resistance associated protein (dMRP; CG6214) was correlated with reduced secretion of the P-glycoprotein substrate daunorubicin. Secretion of the organic cation quinacrine was unaffected by reduced expression of OATP, dMRP, or a multidrug efflux transporter (MET; CG30344). The results highlight the difficulties of assigning a rate-limiting role in transport of a specific OA to a single membrane transporter.