When mustard gas reacts with DNA one of the products consists of two guanines linked by a mustard molecule (GMG). This compound can arise from a pair of guanines lying in opposite strands of the DNA molecule and in this form is the basis of the cross-linking effect of mustard gas on the DNA molecule. The present study shows that GMG arises also in significant amounts from the reaction of two guanines that lie in the same strand. Using DNA isolated from mustard gas treated mammalian cells or alkylated in vitro, the number of GMG's per unit length of DNA was determined. The extent of renaturability which measures the degree of cross-linking was also determined. The number of GMG's exceeded the number of cross-links. When alkylated DNA was separated into a renaturable (cross-linked) fraction and one that contained single strands, analysis showed that both fractions contained GMG.