The authors have recently shown that immunophotodetection of human colon carcinomas in nude mice and in patients is possible by using anti-carcinoembryonic antigen monoclonal antibodies (MAb) coupled to fluorescein. The most common clin. application of photodiagnosis has been for the detection of squamous cell carcinomas (SCC) in the upper respiratory tract, but the free dyes used to have a poor tumor selectivity. The authors selected the known MAb E48 directed against SCC and coupled it to a fluorescent dye: indopentamethinecyanin (indocyanin). This dye has an advantage over fluorescein in that it emits a more penetrating fluorescent red signal at 667 nm after excitation with a laser ray of 640 nm. In vitro, a conjugate with an indocyanin:MAb molar ratio of 2, and an addnl. trace labeling with 125I, showed more than 80% of binding to cells from the SCC line A431. In vivo, when injected i.v.into nude mice bearing xenografts of the same carcinoma line, the MAb E48-(indocyanin)2 conjugate was almost as efficient as the unconjugated MAb E48 in terms of specific tumor localization: 15% of the injected dose per g of tumor at 24 h after injection and a tumor:overall normal tissue ratio of 6-8. There was no selective tumor localization of an irrelevant IgG1-(indocyanin)2 conjugate. Immunophotodetection of the s.c. SCC xenografts on mice given injections of 100 mg of MAb E48-(indocyanin)2 conjugate (representing 1 mg of indocyanin) was performed at 24 h. Upon laser irradn., clearly detectable red fluorescence from the indocyanin-MAb conjugate was obsd. specifically in the SCC xenografts across the mouse skin. In comparison, injection of 100 mg of a MAb E48 coupled to 2 mg of fluorescein gave a specific green fluorescence signal in the tumor xenografts, which was detectable, however, only after removing the mouse skin. Injection i.v. of a 15 times higher amt. of free indocyanin (15 mg) gave a diffuse red fluorescence signal all over the mouse body with no definite increase in intensity in the tumor, indicating a lack of tumor selectivity of the free dye. The results demonstrate the possibility of broadening and improving the efficiency of tumor immunophotodiagnosis by coupling to a MAb directed against SCC, a fluorescent dye absorbing and emitting at higher wavelength than fluorescein, and thus having deeper tissue penetration and lower tissue autofluorescence. Such a demonstration opens the way to a new form of clin. immunophotodiagnosis and possibly to the development of a more specific approach to phototherapy of early bronchial carcinomas.