The use of cholesterol-based cationic lipids and the ability of glucocorticoids to reduce local inflammatory response to lipoplexes motivated an investigation of structure-activity relationships for cationic steroids. A one-step synthetic scheme using iminothiolane was developed to link spermine to the 21-OH position of steroids via an amidine linkage. Five steroids (cortisol, dexamethasone, corticosterone, 11-deoxycortisol, and 11-deoxycorticosterone) with increasing hydrophobicity of the parent steroid (Log Pster from 1.51 to 3.01) were conjugated with spermine, formulated with dioleoylphosphatidylethanolamine (DOPE) at DOPE : steroid mole ratios (R) of R = 0.5 to 2, and then complexed with 1 µg enhanced green fluorescent protein (EGFP) plasmid DNA at charge ratios (CR) = 2 to 24 amines per phosphate (0.5 to 6 steroids per phosphate). The resulting 105 different formulations of the cationic steroid series were used to lipofect bovine aortic endothelial cells. Transgene expression data at either 24 or 48 h post-lipofection for all formulations was collapsed onto master curves when plotted against a single empirical dimensionless parameter, the lipofection index (LI) = CR (Log Pliposome)(Log Pster/|ΔLog P|) [R/(R + 1)] where ΔLog P = Log PDOPE− Log Pster and Log Pliposome is a mole-weighted average of the DOPE/cationic steroid liposome hydrophobicity. For 7 < LI < 29, the EGFP expression at 24 or 48 h post-lipofection increased linearly with LI (EGFP ∼0 for LI < 7), but did not increase further for LI > 29, thus providing a predictive design rule based on Log P of the hydrophobic moiety of the cationic steroid lipid. Copyright © 2007 John Wiley & Sons, Ltd.