The hemodynamic effects of various numbers of colored nonradioactive microspheres (CMS) and those of accumulation of CMS caused by multiple sequential injection were evaluated in 51 Sprague-Dawley male rats. CMS (15 microns) were injected into the left atrium. Regional blood flow and cardiac output were evaluated using the reference blood sample technique. Ficoll-70 was given after each blood sample withdrawal as a fluid replacement. A bolus injection of < or = 1,000,000 CMS caused no significant hemodynamic disturbances. Amounts of 500,000 CMS can be repeatedly injected up to four times (cumulative dose of 2,000,000 CMS) without producing any adverse hemodynamic effects. The values of cardiac output obtained with the CMS technique were correlated well (r = 0.971, P < 0.0001) with those obtained with electromagnetic flow probes. An excellent reproducibility of organ blood flow was observed after four sequential injections of 500,000 CMS. This study establishes the limits of CMS that can be injected into the rat without inducing hemodynamic changes and also suggests that the CMS technique can be employed to evaluate cardiac output and regional blood flow precisely and repeatedly.