We describe two near-infrared fluorescent squaraine dyes (Sq635 and Sq660), their spectra, their covalent linkage to proteins, and their use as donor and acceptor, respectively, in a fluorescence resonance energy transfer (FRET) immunoassay based on the use of red lasers. The dyes show quantum yields of around 10% in the free form and up to 68% when bound to proteins. If converted into their N-hydroxysuccinimide esters, they can be linked to free amino groups of proteins. To improve water solubility, two sulfo groups were introduced. The emission spectrum of Sq635 overlaps the absorption spectrum of Sq660, a fact that makes them a useful pair of dyes for use in FRET immunoassay which is demonstrated for human serum albumin/anti-human serum albumin. (C) 2000 Academic Press.