Background <p>Isoxazolines, including sarolaner, lotilaner, afoxolaner and fluralaner, are a class of ectoparasiticides that target gamma-aminobutyric acid receptors (GABARs) in insects and acari. However, their potential action on mammalian GABARs has not been extensively compared.</p> Methods <p>This study investigated the inhibitory effects of these isoxazolines on human and canine GABARs expressed in <i>Xenopus</i> oocytes. Eleven functional GABAR subunit combinations from human and canine isoforms were successfully cloned and expressed. Two-electrode voltage-clamp measurements were performed to determine the inhibitory effects of the isoxazolines.</p> Results <p>Sarolaner, afoxolaner and fluralaner exhibited partial to high inhibition of human and canine GABARs, with fluralaner showing the lowest half-maximal inhibitory concentration (IC₅₀) values (1.9–13&#xa0;µM). In contrast, lotilaner had little or no inhibitory effect, with IC₅₀ values &gt; 30&#xa0;µM for both human and canine GABARs.</p> Conclusions <p>While neurological adverse events have been reported in dogs, particularly in breeds with the multidrug resistance 1 (MDR1) gene mutation, this study suggests that direct inhibition of canine GABARs may not be the primary cause. However, the interpretation of these results represents a challenge, and a direct correlation with documented cases of adverse events remains difficult. Further research is needed to understand the exposure of mammalian GABARs to isoxazolines and the analog-specific safety risks associated with the observed in vitro receptor activities.</p> Graphical Abstract <p></p>