Aequoreu green fluorescent protein (GFP) has been expressed in a variety of cell lines and host organisms. A recent report (Heim et d.: Proc Natl mented that a GFP mutant with a single amino acid substitution (tyrosine 66 to histidine; Y66H-GFP) elicits altered spectral properties. Whereas wildtype GFP emits with a maximum at approximately 509 n m (green fluorescence), Y66H-GFP fluoresces with a maximum at approximately 448 nm (blue fluorescence). In this study we employed available argon and krypton ion laser lines to investigate the impact of laser excitation wavelength on the detection of Y66H-GFP by flow cytometry. Using transiently transfected 293 cells, a cellular subpopulation with elevated blue fluorescence was detectable with excitation at 407 nm, but not with ultraviolet A-d Sci USA 91:12501-12504, 1994) has dom-(W), 458 nm, or 488 nm excitation. The blue-fluorescing cells were further documented to express Y66H-GFP by immunoblot analysis of sorted cells. Finally, we demonstrated the simultaneous analysis of both wild-type and Y66H-GFP in cotransfected 293 cells using 407 nm excitation while collecting blue fluorescence at 460 f 20 nm (Y66H-GFP) and green fluorescence at 525 f 25 n m (wild-type GFP). These studies illustrate the potential for assessing differential gene expression by simultaneously analyzing multiple GFP species with multipatameter flow cytometry. o 1996 w i l e y -ฤฉ , I~C.