A disadvantage of nonradioactive microsphere techniques is that the processing of samples is time-consuming and complex. We developed and validated a simplified processing method for the fluorescent microsphere (FM) technique. In seven anesthetized dogs with coronary artery stenosis up to six different FM and five different radioactivity labeled microspheres (RM) were injected. Two FM and two RM labels were injected simultaneously to enable inter- and intramethod comparison. After gamma-counting samples of blood, myocardium (n = 168), and other organs (n = 59) were digested in test tubes with 2 N ethanolic KOH (60 degrees C, 48 h), microspheres were sedimented by centrifugation, dye was extracted in the same tube, and fluorescence was measured. With this processing method, recovery of FM was approximately 100%. Good correlations for inter- and intramethod comparisons were found [r = 0.985 +/- 0.01 (mean +/- SD)]. The lower intermethod correlation for blue microspheres (r = 0.958) indicates that the use of this label is less desirable. RM and FM endocardial-to-epicardial blood flow ratios correlated well (r = 0.974). With this one-vessel centrifugal sedimentation method and at least five fluorescently labeled microspheres, blood flow can be reliably measured in various organs, including ischemic myocardium.