We have developed a simple and rapid method for the determination of retroviral titer by G418 selection which is both accurate and reproducible. NIH3T3 cells were transduced with recombinant retroviral vectors harboring the neo gene and selected in the presence of the antibiotic G418 for 3 days. Cells were then trypsinized, harvested, and counted on a hemacytometer. The control NIH3T3 cells were completely dead 3 days after treatment with G418, while cells transduced with retroviral vectors containing the neo gene produced an average of 15 progeny cells per colony. To estimate viral titer, therefore, the total number of trypsinized G418-resistant cells was divided by 15. Retroviral titers measured by this method did not differ significantly from those determined by the conventional method based on counts of G418-resistant colonies 10-14 days after G418 selection. The method worked well with various retroviral constructs and its efficacy was not influenced by cDNA insert. This method not only reduces the amount of work but also shortens the time needed for determining viral titer.