Perfusion, the rate at which blood in tissue is replenished at the capillary level, is a primary factor in the transport of heat, drugs, oxygen and nutrients. While there have been many measurement techniques proposed, most do not lend themselves to routine, continuous and real-time use. A minimally invasive probe, called the thermal diffusion probe (TDP), which uses a self-heated thermistor to measure absolute perfusion continuously and in real time, was validated at low flows with the microsphere technique. In 27 rabbits, simultaneous TDP measurements were made in liver from 0 to 60 ml min-1 100 g-1. The TDP perfusion correlated well with the microspheres (R2 = 0.898) and the agreement between techniques is very good with a slope close to unity (0.921) and an intercept close to zero (0.566 ml min-1 100 g-1). Variability between the two techniques was primarily due to the sampling error from the microsphere 'snap shot' of periodic blood flow when compared with the continuous TDP perfusion measurement. The ability to quantify local perfusion continuously and in real time may have a profound impact on patient management in a number of clinical areas such as organ transplantation, neurosurgery, oncology and others, in which quantitative knowledge of perfusion is of value.