The high sensitivity of fluorescent reporters offers an opportunity to analytically probe the biochemistry of in vivo receptor systems with low target tissue concentration. We investigated the ability of an optical imaging system to acquire adequate signal for in vivo measurement of receptor biochemistry. The imaging system consisted of a small animal optical imager operating in the time domain (TD) and a fluorescent-labeled diagnostic probe of known receptor-binding properties. Optical imaging of mice (n = 4) using the targeted probe, Cy5.5-DTPA-galactosyl-dextran (2.2 Cy5.5, 4 DTPA, 68 galactose units per dextran, 124 kDa, 24 nmol/kg), demonstrated blood clearance and hepatic uptake. The mean and standard deviation for the time to reach 90% of the peak liver intensity were 15.4 +/- 1.6 min. Typical fluorescent intensities within a 10-pixel region-of-interest from a 30-s image acquired 30 min postinjection were in excess of 2.5 million counts. The nontargeted agent (Cy5.5-DTPA-dextran) did not demonstrate (n = 4) hepatic uptake. This uptake pattern was duplicated by nuclear imaging of rabbits using (99m)Tc-labeled Cy5.5-DTPA-galactosyl-dextran and Cy5.5-DTPA-dextran. This study demonstrated the feasibility of optically labeling a receptor-binding diagnostic probe and imaging in the TD with sufficient sensitivity and temporal resolution for pharmacokinetic analysis.