A principal protective component of the mammalian blood-brain barrier (BBB) is the high expression of the multidrug efflux transporters P-glycoprotein (P-gp, encoded byABCB1) and ABCG2 (encoded byABCG2) on the luminal surface of endothelial cells. The zebrafish P-gp homolog Abcb4 is expressed at the BBB and phenocopies P-gp. Comparatively little is known about the four zebrafish homologs of the humanABCG2gene:abcg2a,abcg2b,abcg2c, andabcg2d. Here we report the functional characterization and brain tissue distribution of zebrafish ABCG2 homologs. To determine substrates of the transporters, we stably expressed each in HEK-293 cells and performed cytotoxicity and fluorescent efflux assays with known ABCG2 substrates. We found Abcg2a had the greatest substrate overlap with ABCG2, and Abcg2d appeared to be the least functionally similar. Using RNAscopein situhybridization we identifiedabcg2aas the only homolog expressed at the adult and larval zebrafish BBB, based on its localization to claudin-5 positive brain vasculature. These results demonstrate the conserved function of zebrafish Abcg2a and suggest that zebrafish may be an appropriate model organism for studying the role of ABCG2 at the BBB.