We have developed a method for measuring regional blood flow by means of fluorescent microspheres in all organs and tissues of the fetal lamb, including brain, heart, lung, liver, gut, spleen, kidney, adrenal, brown fat, skin, muscle, bone, and placenta. Five different fluorescent-labeled microspheres were used: blue (B), yellow-green (Y), orange (O), red (R), and crimson (C). An automated, 96-well microplate fluorescent reader (bottom reading) was chosen for the assay because of the rapidity and high throughput that it offers. Tissue samples were digested by 4 M ethanolic KOH. The sedimentation method and dye extraction with Cellosolve acetate, as previously reported by others, were used for the sample processing. The bones were crushed and allowed to directly soak in Cellosolve acetate to extract the dye. The relationship between microsphere number and fluorescent intensity was linear over a broad range of microsphere numbers (80 - 20 000/mL). The coefficients of variation of within-run and between-run precision were 3.39 +/- 1.10% and 4.54 +/- 1.10%, respectively. Recovery of microspheres from tissues and blood averaged 94.3 +/- 2.5% and was not dependent on microsphere number. The spillover of the fluorescent signals into adjacent colors was 4.0 +/- 0.1% for O to Y, 8.1 +/- 0.4% for O to R, and 9.1 +/- 0.5% for R to C, and these values were constant over a wide range in concentrations of the microsphere pairs. No evidence was obtained for quenching of the emission of one fluorophore via photon absorption by another fluorophore. The measurements of regional blood flow obtained with fluorescent microspheres in three chronically instrumented fetal lambs at similar to 140 days gestation were similar to the flow estimates obtained using radioactive microspheres in four other fetal lambs at the same gestational age. The fluorescent method is thus a viable alternative to the radioactive technique for the measurement of regional blood flow to all fetal organs and tissues, particularly when an automated fluorescent microplate reader is employed to reduce analysis time.