Molecular imaging of cell surface receptors can potentially diagnose tumors based on their distinct expression profiles. Using multifilter spectrally resolved optical imaging with three fluorescently labeled antibodies, we simultaneously imaged three different cell surface receptors to distinguish tumor types noninvasively. We selected tumors overexpressing different subtypes of EGFR receptor: HER-1 (A431) and HER-2 (NIH3T3/HER2(+)), or interleukin-2 receptor alpha-subunit receptor (IL-2Ralpha; SP2/Tac). After tumor establishment, a cocktail of three fluorescently labeled monoclonal antibodies was injected: cetuximab-Cy5 (targetingHER-1), trastuzumab-Cy7(HER-2),anddaclizumab-AlexaFluor-700 (IL-2Ra). Optical fluorescence imaging was performed after 24 hours with both a red filter set and three successive filter sets (yellow, red, and deep red). Spectrally resolved imaging of 10 mice clearly distinguished A431, NIH3T3/HER2(+), and SP2-Tac tumors based on their distinct optical spectra. Three-filter sets significantly increased the signal-to-background ratio compared to a single-filter set by reducing the background signal, thus significantly improving the differentiation of each of the receptors targeted (P < .022). In conclusion, following multifilter spectrally resolved imaging, different tumor types can be simultaneously distinguished and diagnosed in vivo. Multiple filter sets increase the signal-to-noise ratio by substantially reducing the background signal, and may allow more optical dyes to be resolved within the narrow limits of the near-infrared spectrum.