Although many known receptors that regulate addiction have been pharmacologically and biochemically well characterized, some orphan receptors with homology to known receptors of abuse (i.e. GPR35) remain uncharacterized. GPR35 is a G-protein coupled receptor, first identified in 1998 after a screen of a human genomic library. More recent RT-PCR studies have now confirmed the presence of GPR35 in dorsal root ganglion, the cerebellum and brain, as well as GPR35b, which was cloned from a human whole brain cDNA library. Thus, GPR35 regulation appears to have profound physiological and pathophysiological implications. We have identified a 3rd antagonist, ML194 that represents a different chemical scaffold with potency (160 nM) and selectivity (>57-fold) for GPR35, but not for the related GPR55 orphan receptor, that is intermediate between the previously reported probes, ML145 (CID2286812) and ML144 (CID1542103). ML194 also does not seem to produce non-specific interference with signaling directly at or downstream of the β-arrestin signaling pathway, so it may serve as an additional tool to delineate the biochemistry of GPR35 as potential therapeutics to selectively target pathways underlying pain and to enhance our understanding of the molecular basis of addiction.