Nuclear import of SV40 large T-antigen (T-ag) is completely dependent on the T-ag nuclear localization sequence (amino acids 126-132), but the rate of nuclear import is greatly increased by the additional presence of the N-terminal flanking sequence (amino acids 111-125), which includes a site for casein kinase II (CKII) (Ser111/112). The role of this site was investigated by site-directed mutagenesis and analysis of the effects on phosphorylation and on nuclear import at the single cell level using microinjection and quantitative fluorescent techniques. Removal of the CKII site either by substitution of S111/112 by nonphosphorylatable amino acid residues, or mutation of the Asp-Asp-Glu113/115 CKII recognition sequence to Asn-Asn-Gln, resulted in nuclear import rates less than 4% wild type, demonstrating that the CKII site was responsible for the enhancement of nuclear import conferred by T-ag amino acids 111-125. The substitution of Asp for Ser112, the serine preferentially phosphorylated by purified CKII, enhanced nuclear import to about 45% maximal wild type rates. It is concluded that negative charge at the CKII site, normally provided by phosphorylation, is mechanistically important for nuclear transport enhancement. There was no evidence or a direct role for phosphatases or dephosphorylation in the transport process.