A rapid screening procedure for restriction fragment length polymorphisms (RFLPs) is reported. DNA from ten individuals is pooled and compared to DNA isolated from a cell line containing a single chromosome 4. This single chromosome-containing line is an obligate hemizygote for chromosome 4 RFLPs so that only one band corresponding to a single allele will appear on a Southern blot. In the pooled DNA sample lane bands corresponding to both alleles will be seen. The technique allows for efficient detection of RFLPs with easier use of large numbers of enzymes. It provides estimates of allele frequencies and disequilibrium. New RFLPs for albumin and alcohol dehydrogenase detected with this technique are described.