We have examined the S1 nuclease sensitivity of supercoiled plasmids harboring the Moloney Murine Leukemia Virus (MoMuLV) long terminal repeat (LTR). S1 sensitivity was found within the LTR enhancer direct repeats. Transformation of E. coli DH5 cells with a construct containing most of the MoMuLV LTR yielded the precise deletion of one direct repeat and loss of S1 sensitivity. The dependence of S1 sensitivity on the presence of both direct repeats, together with the exact excision of one direct repeat by E. coli, suggests the presence of slipped DNA within the enhancer. Such structures may represent targets for effector proteins which mediate vital functions during viral propagation.