We have examined the effect of protease inhibitors on radiation-induced DNA amplification in vitro using an SV40-transformed Chinese hamster embryo cell line. DNA from cells irradiated with X-rays (10 Gy) or ultraviolet radiation (8 J/m2) resulted in an 8-fold or greater amplification of SV40 sequences compared with unirradiated cells. Addition of antipain or the soybean-derived Bowman-Birk protease inhibitor (BBI) to the culture medium, either 75 min after X-irradiation or within 15 min after ultraviolet irradiation, resulted in a reduction of amplification levels. BBI was more effective than antipain at an equal concentration. These results suggest that a protease may be involved in DNA amplification.