The interactions between calf thymus DNA and a pyrene-labeled gemini surfactant termed PyO-3-12 were investigated by using dynamic light scattering (DLS), transmission electron microscopy (TEM), and pyrene excimer formation (PEF) between an excited and a ground-state pyrene. PyO-3-12 was prepared with two dimethylammonium bromide headgroups linked with a propyl spacer, with one ammonium bearing a dodecyl tail and the other bearing a 1-pyrenemethoxyhexyl pendent. The size and electrostatic properties of the colloidal species were characterized by DLS, and TEM was used to describe the overall dimensions and morphologies of the aggregates. PEF enabled the study of PyO-3-12 at the molecular level. The association of PyO-3-12 and DNA was studied in detail for two specific PyO-3-12 concentrations of 16 and 56 μM using a (∓) ratio equal to the [DNA]/[PyO-3-12] ratio with [DNA] expressed in terms of DNA base pairs, yielding a (∓) ratio ranging from 0.1 to 10. An increase in PyO-3-12 association upon adding DNA was detected through an increase in PEF. Analysis of the fluorescence decays indicated a progressive binding of PyO-3-12 to DNA until the equicharge point was reached after which all PyO-3-12 surfactants were bound to DNA. The formation of large colloidal aggregates was observed by DLS, which showed a spike in the hydrodynamic diameter (