Cultured endothelial cells of the human retina and choroid demonstrate distinct patterns of gene expression. We hypothesized that differential gene expression reflected differences in the interactions of transcription factors and respective cis-regulatory motifs(s) in these two emdothelial cell subpopulations, recognizing that motifs often exist as modules. We tested this hypothesis in silico by using TRANSFAC Professional and CisModule to identify cis-regulatory motifs and modules in genes that were differentially expressed by human retinal versus choroidal endothelial cells, as identified by analysis of a microarray data set. Motifs corresponding to eight transcription factors were significantly (p < 0.05) differentially abundant in genes that were relatively highly expressed in retinal (i.e., GCCR, HMGIY, HSF1, p53, VDR) or choroidal (i.e., E2F, YY1, ZF5) endothelial cells. Predicted cis-regulatory modules were quite different for these two groups of genes. Our findings raise the possibility of exploiting specific cis-regulatory motifs to target therapy at the ocular endothelial cells subtypes responsible for neovascular age-related macular degeneration or proliferative diabetic retinopathy.