The small proline-rich protein genes ( SPRRs ) code for precursors of the cornified cell envelope, and are specifically expressed during keratinocyte terminal differentiation. The single intron of SPRR2A enhanced the activity of the SPRR2A promoter in transient transfection assays. This enhancement was position dependent, and did not function in combination with a heterologous promoter, indicating that the intron does not contain a classical enhancer, and that the enhancement was not due to the splicing reaction per se. Mild DNAse-I digestion of nuclei showed the SPRR2 genes to be tightly associated with the nuclear matrix, in contrast to the other cornified envelope precursor genes mapping to the same chromosomal location (epidermal differentiation complex). In vitro binding studies indicated that both the proximal promoter and the intron of SPRR2A are required for optimal association of this gene with nuclear matrices. Neither nuclear matrix association nor the relative transcriptional enhancement by the intron changed during keratinocyte differentiation. Apparently, the association of the SPRR2A gene with the nuclear matrix results in a general, differentiation-independent enhancement of gene expression.