Fluorescence microphotolysis was employed to measure in single living cells the kinetics of nucleocytoplasmic transport and the coefficients of intracellular diffusional mobility for the nuclear non-chromosomal protein nucleoplasmin. Nucleoplasmin was isolated from Xenopus ovary and labeled fluorescently. By injection into Xenopus oocytes it was ascertained that fluorescent labeling did not interfere with normal nuclear accumulation. Upon injection into the cytoplasm of various mammalian cell types nucleoplasmin was rapidly taken up by the nucleus. In rat hepatoma cells the half-time of nuclear uptake was approx. 5 min at 37 degrees C; the nucleocytoplasmic equilibrium concentration ratio had a maximum of 6.5 +/- 1.4 and depended on the injected amount. Upon co-injection of ATPases or reduction of temperature to 10 degrees C a nucleocytoplasmic equilization but no nuclear accumulation was observed. Equilization was fast (time constant 65 s at 23 degrees C), similar to that of 10-kDa dextran permeating the nuclear envelope by simple diffusion through functional pores. Nucleoplasmin (160 kDa), however, is too large to permeate passively the nuclear envelope, which is apparent from the fact that its tryptic 'core' fragment (100 kDa) could not permeate the nuclear envelope. On the other hand, a large fluorescent protein, phycoerythrin (240 kDa), was targeted to the nucleus by conjugation with nucleoplasmin. In the nucleus-to-cytoplasm direction the nuclear envelope was completely impermeable to nucleoplasmin, independently of temperature or ATP depletion. Nucleoplasmin, its core fragment, phycoerythrin and the phycoerythrin-nucleoplasmin conjugate were mobile in both cytoplasm and nucleus.