Measurement of regional organ blood flow by means of fluorescent microspheres (FM) is an accepted method. However, determination of regional portal blood flow (RPBF) can not be performed by microspheres due to the entrapment of the spheres in the upstream capillary bed of the splanchnic organs. We hypothesized that an adequate experimental setting would enable us to measure RPBF by means of FM and to analyze it's distribution within the pig liver. A mixing chamber for the injection of FM was developed and it's capability to distribute FM homogeneously in the blood was evaluated in vitro. The chamber was implanted into the portal vein of six anaesthetized pigs (BW 23.5 ± 2.9 kg). Three consecutive, simultaneous injections of FM of two different colors into the chamber were performed. Reference portal blood samples were collected by means of a Harvard-pump. At the end of the experiment the liver was explanted and fixed in formalin prior to dissection. FM were isolated from the tissue samples by an automated process and fluorescence intensity was determined. Comparison of 5,458 single RPBF-values determined by simultaneously injected FM revealed good agreement (bias 2.5%, precision 12.7%) and high correlation (r = 0.97, r² = 0,95, slope = 1.04, intercept = 0.05). Median RPBF was 1.07 ± 0.78 ml/min/g. Allocation of the blood flow values to the anatomical regions of the liver revealed a significant higher RPBF (p=0.01) in the liver tissue located close to the diaphragm compared to the rest of the organ and a significant lower RPBF (p=0.01) in the left liver lobe as compared to the median and right lobe. The results show that the model presented makes it possible to measure RPBF by means of FM reliably and that RPBF is distributed heterogeneously in the porcine liver.