Recent studies of the sieving of serum albumin in the rat kidney using a two-photon microscope suggested that the glomerular sieving coefficient (GSC) of albumin is 0.034, much higher than earlier micropuncture determinations. In the present study, we critically evaluated the use of the two-photon microscope to measure the GSC of albumin in the Munich-Wistar rat in vivo. The albumin GSC averaged 0.004 (SD 0.004), n = 34 glomeruli, when determined with a Zeiss two-photon microscope system and 0.002 (SD 0.002), n = 5, when determined with an Olympus two-photon microscope system. These values are close to the lower limit of detection of GSC, which we estimate to be about 0.001-0.003. We identified several factors that were likely responsible for the higher albumin GSCs reported earlier using two-photon microscopy. These include animal conditions (i.e. low glomerular filtration rate) and failure to recognize the role of out-of-focus fluorescence in contaminating the fluorescence signal from the urinary space of Bowman's capsule. We observed that hypothermia plus dehydration or a low blood pressure led to an increased albumin GSC. High levels of illumination (high laser outputs) resulted in a falsely elevated albumin GSC. Use of external, instead of internal, photodetectors resulted in an exaggerated albumin GSC because of greater collection of out-of-focus fluorescence. In conclusion, the albumin concentration in the glomerular filtrate of the normal rat, determined by two-photon microscopy, is exceedingly low (5-10 mg/dL).