Regulatory elements of the osteopontin (opn) gene are attractive candidates for expressiontargeted gene therapy because numerous malignant cancers are marked by opn overexpression. The maximum opn promoter (Popn)-driven reporter intensity obtained for tested cancer cell lines was as strong (102.69%) as positive-control transfections. At the same time, Popn-driven reporter expression was reduced by ~90% in non-cancer cell lineages. Deletion analysis of the -922 bp region opn promoter did not confirm published reports of a repressor area within 922 bases upstream of the transcriptional start site. Further enhancements to targeting and expression were obtained through incorporation of single-nucleotide polymorphisms (SNPs) into the promoter sequence. It was found that the SNPs -443C, -155GG, -66T led to increased Popn-driven transfection in cancer cells (fold increase of 1.23 ~ 3.48), with a concomitant decrease in reporter expression in normal controls (fold change of 0.69). Further investigations to confirm a correlation between endogenous opn mRNA levels and Popn-driven reporter expression produced a surprising lack of correlation (R2=0.24). However, taking into account opn mRNA splicing variants showed a strong negative correlation between mRNA levels of the variant opn-a and P opn-driven transgene activity (R2=0.95). These data have implications on how future searches for expression-targeting promoters should be conducted.