Palonosetron hydrochloride is a relatively long-acting selective 5-HT3 receptor antagonist that has been approved for the prevention of chemotherapy-induced nausea and vomiting and is being evaluated for the prevention of postoperative nausea and vomiting. The objective of this study was to evaluate the physical and chemical stability of palonosetron hydrochloride 50 mcg/mL when mixed with gentamicin sulfate 5 mg/mL, metronidazole 5 mg/mL, or vancomycin hydrochloride 5 mg/mL during simulated Y-site administration. Duplicate samples of palonosetron hydrochloride with each of the anti-infectives were tested. Samples were stored and evaluated for up to 4 hours at room temperature. Physical stability was assessed by turbidimetric and particulate measurements and visual inspection. Chemical stability was assessed by high-performance liquid chromatography. All of the admixtures were clear and colorless when viewed in normal fluorescent room light and when viewed with a Tyndall beam. Measured turbidity and particulate content were low initially and remained low throughout the study. The drug concentration was unchanged in all of the samples tesed. Palonosetron hydrochloride is physically and chemically stable with gentamicin sulfate, metronidazole, or vancomycin hydrochloride at the concentrations tested during simulated Y-site administration over 4 hours at ambient room temperature.