Superhelical simian virus 40 (SV40) DNA I can be modified with N-cyclohexyl-N'-beta-(4 methylmorpholinium)ethylcarbodiimide (CMC). The reaction produces an increase in the sedimentation velocity of DNA I from 21 to 22.5S and a decrease in its buoyant density in CsCl from 1.694 to 1.688. A comparable shift in buoyant density is observed in a saturated ethidium bromide-cesium chloride gradient where form II, which has been exposed to CMC, shows no shift. The CsCl-buoyant density data allows us to estimate that 108 mol of CMC are bound per mol of SV40 DNA I. In the subsequent paper an alternative procedure has been used to locate CMC sites, and the extent of the regions available to bind CMC have been measured.