The goal of this study is to elucidate the mechanism by which nitrogen mustard, a difunctional alkylating agent, inhibits DNA template activity for RNA synthesis. This inhibition could be due not only monofunctional alkylations, known to hinder RNA synthesis, but also to interand intrastrand bridges formed by difunctional alkylation. DNA from bacteriophage T, and Escherichia coli RNA polymerase provide a well-characterized system for RNA synthesis in vitro. Only 20°/, of the T, genome is transcribed by E . coli RNA polymerase and hence only those alkylations falling in this region could influence RNA synthesis.Three types of experiments were carried out. T, DNA was alkylated by treatment with varying concentrations of nitrogen mustard. This alkylated DNA was used as template for RNA synthesis; the extent of interstrand crosslinking in the DNA was compared with its loss of template activity. Interstrand crosslinks were gradually eliminated from alkylated T, DNA by incubation, under sterile conditions, for variable times at 37 "C; these DNAs were then used as templates for RNA synthesis in vitro; the antibiotic, rifampicin, was included in the synthesis medium to eliminate non-specific chain initiation. T, DNA was alkylated with radioactive nitrogen mustard to calculate the extent of alkylation a t the dose which reduces activity to 3701, of the control.Template activity decreases with increasing doses of nitrogen mustard ; however, the fraction of DNA molecules remaining without interstrand crosslinks decreases much more rapidly. Since only 20°/, of T, DNA is transcribed by E. coli RNA polymerase, we examined the relation between the fraction of molecules bearing one interstrand crosslink in this region and the inhibition of RNA synthesis. The inhibition is significantly greater ; interstrand orosslinks could, a t the most, explain only one third of the activity loss.As interstrand crosslinks disappear from alkylated T, DNA, its template activity, in the presence of rifampicin, decreases. A significant but smaller loss of activity (39°/0 after 72 h of incubation) is observed in the non-alkylated control DNA. When RNA synthesis is carried out in the absence of rifampicin, the activity losses of the control and of the alkylated sample are entirely comparable. Had interstrand crosslinks been responsible for the initial loss of activity after alkylation, their disappearance should have caused a reactivation ; had they been without influence, activity would have been expected to remain constant. We tentatively interpret our result to indicate that other phenomena, i.e. depurination and single strand breakage, which occur during the incubation of alkylated DNA, are responsible for the loss of activity; the possible effect of interstrand crosslinks may thus be masked.At the concentration of nitrogen mustard which reduces DNA template activity to 37O/, of the control, T, DNA is alkylated to the extent of 90 alkyl groups per molecule. This dose is about 9 times less than that necessary with methylation or ethylation of T,...