The β-cell loss seen in diabetes mellitus could be monitored clinically by positron emission tomography (PET) if imaging agents were sufficiently specific for β-cells to overcome the high ratio of non-β-cell to β-cell tissue in pancreas. In this report, we present a screening assay for identifying β-cell-specific compounds that is based on the relative accumulation and retention by islet, INS-1, and exocrine (PANC-1) cells of candidate molecules. Molecules thought to have a high affinity for β-cells were tested and included glibenclamide, tolbutamide, serotonin, l-DOPA, dopamine, nicotinamide, fluorodeoxyglucose, and fluorodithizone. Glibenclamide and fluorodithizone were the most specific, but the specificity ratios fell well below those needed to attain robust signal to background ratio as a PET imaging agent for quantifying β-cell mass. In vivo tests of the biodistribution of glibenclamide and fluorodithizone in rats indicated that the compounds were not specifically associated with pancreas, bearing out the predictions of the in vitro screen.