Telavancin is a semisynthetic lipoglycopeptide with a dual mechanism of action against Gram-positive pathogens. Two brief reports have suggested potential cross-reactivity of telavancin with the vancomycin particle-enhanced turbidometric immunoassay (PETIA). The purpose of this study was to evaluate several commercially available vancomycin immunoassays (fluorescence polarization [FPIA], enzyme-multiplied immunoassays [EMIT], PETIA, and chemiluminescent immunoassay [CMIA]) for cross-reactivity with telavancin. Seven sites were selected to analyze serum samples for vancomycin. Each site received a set of samples (n ؍ 18) which combined drug-free serum with telavancin, 7-OH telavancin metabolite, or vancomycin. Immunoassays demonstrating potential cross-reactivity were further evaluated by sending a duplicate sample set to multiple laboratories. Cross-reactivity was defined as the percent theoretical concentration (reported concentration/theoretical concentration ؋ 100). No cross-reactivity was seen with FPIA or EMIT. Within the theoretical concentration range of 5 to 120 g/ml of telavancin, the Synchron PETIA system reported vancomycin concentrations ranging from 4.7 to 54.2 g/ml compared to vancomycin concentrations from 1.1 to 5.6 g/ml for the Vista PETIA system. The Architect CMIA system reported vancomycin concentrations in the range of 0.27 to 0.97 g/ml, whereas Advia Centaur XP CMIA reported vancomycin concentrations between 1.6 and 31.6 g/ ml. The Architect CMIA immunoassay had the lowest percent cross-reactivity (0.8 to 5.4%), while the Synchron PETIA immunoassay demonstrated the highest percent cross-reactivity (45.2 to 53.8%). Telavancin samples measured by liquid chromatography-mass spectroscopy were within 93.9 to 122% of theoretical concentrations. Vancomycin concentrations were not measured in any 7-OH telavancin-spiked sample. Vancomycin concentrations measured by liquid chromatography-mass spectroscopy were within 57.2 to 113% of theoretical concentrations. PETIA and CMIA measured vancomycin concentrations in telavancinspiked samples. Significant variability in percent cross-reactivity was observed for each platform regardless of immunoassay method.