In this study the technique of laser-Doppler flowmetry was evaluated for the measurement of tissue blood flow by comparing laser-Doppler flow (LDF) signal in the renal cortex, gracilis muscle, and cremaster muscle of anesthetized rats to whole-organ blood flow measured with an electromagnetic flowmeter or radioactive microspheres. In vitro, LDF signal was closely correlated (r = 0.99) to changes in erythrocyte velocity generated with a rotating wheel. Although individual LDF readings varied in situ, mean LDF signal calculated from multiple readings on the tissue surface were significantly correlated (r = 0.74-0.95) with tissue blood flows measured at various perfusion pressures. However, significant differences in the slope of the LDF signal vs. blood flow relationship were observed in different tissues and with different methods of measurement in the same tissue. This study indicates that mean laser-Doppler flow signal provides a good estimate of tissue blood flow, provided a sufficient number of points is scanned. However, there appears to be no universal calibration factor for the method.