Autoradiographic measurement of local cerebral blood flow (CBF) with [14C]iodoantipyrine (IAP) is limited in mice by the difficulty in cannulating vessels and the blood loss for repeated blood sampling. The authors modified and validated the method to measure local CBF with [14C]IAP in mice by combining intraperitoneal tracer application with a single blood sampling from the heart at the end of the experiment. Experiments were carried out in male SV129 mice under halothane anesthesia. After intraperitoneal administration of 15 microCi [14C]IAP, arterial blood samples were collected repeatedly and anesthetized animals were immersed in liquid nitrogen. In addition, frozen blood from the heart was sampled to obtain the final blood [14C]radioactivity. Correlation analysis between the sampling time and [14C]radioactivity of the arterial blood revealed a highly significant linear relationship (P < 0.001, r = 0.978) and a lag time of the [14C]tracer in arterial blood of 3.3 +/- 0.6 seconds. [14C]radioactivity of the final arterial blood sample (444 +/- 264 nCi/mL) was almost equal to that of the heart blood (454 +/- 242 nCi/mL), and the absolute difference in each animal was 3.3 +/- 4.2% (mean +/- SD). The convolution integrals for the CBF calculation were determined either by integrating the radioactivity of individual arterial blood samples or by assuming a linear rise from [14C]tracer lag time after intraperitoneal [14C]IAP injection to the value measured in the blood sample from the frozen heart. Regional flow values calculated by the two methods differed by less than 11% (not significant). This method allows the quantitative measurement of local CBF in anesthetized mice without any vessel catheterization and will make mutant mice a more powerful tool to elucidate the molecular mechanisms of brain injuries by combining flow studies with molecular-biological methods.