The rare fragile site at 16q22 was experimentally induced in lymphocyte cultures with various AT-specific, non-intercalating DNA-ligands. The optimum conditions for the induction of fra(16)(q22) were determined. The best expression of fra(16)(q22) was found with the aromatic diamidine berenil which is recommended for further studies on this fragile site. The results indicate that fra(16)(q22) is a region with AT-rich, late replicating DNA. The simultaneous treatment of lymphocytes with berenil and aphidicolin (inhibitor of DNA polymerase alpha) induces both the rare fra(16)(q22) and the common fra(16)(q23) within the same chromosome. A population study on 350 unselected individuals showed that fra(16)(q22) is the most common of all rare autosomal fragile sites in man. The frequency of individuals heterozygous for fra(16)(q22) is 5.1%, no homozygosity for fra(16)(q22) was detected. Statistical analysis indicates that the population is in Hardy-Weinberg equilibrium with respect to the fragile and non-fragile chromosomes 16.