Fluorescence microphotolysis was used to measure nucleocytoplasmic flux in single rat hepatocytes for a series of dextrans ranging in molecular mass from 3 to 150 kd. The cytoplasmic translational diffusion coefficient DC and the nucleoplasmic diffusion coefficient DN of a 62-kd dextran were also determined. DC was approximately 2 X 10(-8) and DN approximately 3 X 10(-8) cm2/s, i.e., 1/20-1/15 of the value in free solution. The mobile fraction amounted to 0.7-0.8 in measurements of both intracellular diffusion and nucleo-cytoplasmic flux. The flux of dextrans from cytoplasm to nucleus depended inversely on molecular mass with an exclusion limit between 17 and 41 kd suggesting that the nuclear envelope has functions of a molecular sieve. Employing the Pappenheimer-Renkin equations, a functional pore radius of 50-56 A was derived. By comparison with recent measurements on isolated liver cell nuclei, large quantitative differences between the intracellularly located and the isolated nucleus were revealed.