The structure of a DNA-dimethyldidodecylammonium bromide (DDAB) film was recently described to undergo a distinctive transition in response to the water content in the surrounding environment.(1) The existence, preparation, and basic properties of DNA-surfactant films have been known in the literature for some time.(2,3) Here, we describe the structural response of DNA-DDAB films to environmental changes, particularly temperature and humidity, in greater detail revealing new structural states. We can direct the lamellar structure of the film into three distinct states double-stranded DNA (dsDNA) paired with an interdigitated bilayer of DDAB (bDDAB), single-stranded DNA (ssDNA) with monolayer of DDAB (mDDAB), and ssDNA with bDDAB. Both temperature and humidity cause the molecules composing the lamellar structure to change reversibly from ssDNA to dsDNA and/or from mDDAB to bDDAB. We found that the structural transition from dsDNA to ssDNA and bDDAB to mDDAB is concerted and follows apparent first-order kinetics. We also found that the double-stranded conformation of DNA in the film can be stabilized with the inclusion of cholesterol even while the DDAB in the film is able to form either a monolayer or bilayer depending on the environmental conditions. Films treated with ethidium bromide prompt switching of dsDNA to ssDNA before bDDAB transitions to mDDAB. Swelling experiments have determined that there is a direct proportionality between the macroscopic increase in volume and the nanoscopic increase in lamellar spacing when a film is allowed to swell in water. Finally, experiments with phosphate-buffered saline (PBS) indicate that the films can disassemble in a simulated biological environment due to screening of their charges by buffer salt. We conclude that the structure of DNA in the film depends on the water content (as measured by the relative humidity) and temperature of the environment, while the state of DDAB depends essentially only on the water content. The structure of the film is quite flexible and can be altered by changing environmental conditions as well as the chemical ingredients. These films will have useful, new applications as responsive materials, for example, in drug and gene delivery.