Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for all pathways of DNA metabolism. To study the function of RPA in living cells the second largest RPA subunit and an N-terminal deletion mutant thereof were fused to green fluorescent protein (GFP; GFP-RPA2 and GFP-RPA2deltaN, respectively) in a controlled, molecular biological way. These proteins were expressed in HeLa cells under the control of the inducible tetracycline expression system. GFP-RPA2 and GFP-RPA2deltaN are predominately nuclear proteins as determined by confocal laser scanning microscopy. Low basal expression of GFP-RPA2deltaN allowed the measurement of kinetic parameters of RPA. Using fluorescence correlation spectroscopy (FCS) two populations--a fast and a slow moving species--were detected in the nucleus and the cytosol of human cells. The translational diffusion rates of these two RPA populations were approximately 15 microm2/s and 1.8 microm2/s. This new finding reveals the existence of different multiprotein and ssDNA-protein complexes of RPA in both cellular compartments and opens the possibility for their analyses.