Confocal microscopy and image analysis were used to compare driving forces, specificity and regulation of transport of the fluorescent organic anion, Texas red (sulforhodamine 101 free acid, TR) in lateral choroid plexus (CP) isolated from rat and an evolutionarily ancient vertebrate, dogfish shark (Squalus Acanthias). CP from both species exhibited concentrative, specific and metabolism-dependent TR transport from bath to subepithelial/vascular space; at steady state, TR accumulation in vascular/subepithelial space was substantially higher than in epithelial cells. In rat CP, steady state TR accumulation in subepithelial/vascular spaces was reduced by Na+-replacement, but not affected by a tenfold increase in buffer K+. In shark CP, Na+-replacement did not alter TR accumulation in either tissue compartment; subepithelial/vascular space levels of TR were reduced in high K+ medium. In both species, steady state TR accumulation was not affected by p-aminohippurate or leukotriene C4, suggesting that neither organic anion transporters (SLC22A family) nor multidrug resistance-associated proteins (ABCC family) contributed. In rat CP, digoxin was without effect, indicating that organic anion transporting polypeptide isoform 2 (Oatp2) was not involved. Several organic anions reduced cellular and subepithelial/ vascular space TR accumulation in both tissues, including estrone sulfate, taurocholate and MK571. In rat CP, TR accumulation in subepithelial/vascular spaces increased with protein kinase A (PKA) activation (forskolin), but was not affected by PKC activation (phorbol ester). In shark, neither PKA nor PKC activation specifically affected TR transport. Thus, rat and dogfish shark CP transport TR, but do so using different basic mechanisms that respond to different regulatory signals.