The purpose of the study is to develop a simple and less invasive method for quantifying regional cerebral blood flow (rCBF) using 99mTc-ECD and SPECT. By employing a microsphere model, a new method to measure rCBF was developed, which required a single arterial or venous sample instead of continuous withdrawal of arterial blood. Using a regression line, the integral of input function of arterial blood from 0 to 30 min was inferred by activity of arterial blood sampled at time t; A(t), by activity multiplied by its octanol extraction rate; AN(t), by activity of venous blood at time t; V(t), and by activity multiplied by its octanol extraction rate; VN(t). The optimum sampling time of arterial or venous blood was examined when mean % error for inference became minimum. Consequently, minimum error of AN(6 min) was 5.5%, A(3 min) was 8.9%, VN(6 min) was 5.9%, and V(20 min) was 10.0%. Quantitative measurement of rCBF using the value of VN(6 min) was performed on other 6 subjects with dementia etc. To validate the method, 133Xe inhalation SPECT studies were also performed on the same subjects. We found a good agreement between them (r = 0.851). The presented one-point sampling methods were simple and less invasive for quantifying rCBF.