The expression of a 3-kilobase genomic rat whey acidic protein (WAP) clone (-949/+2020) in transgenic mice has been demonstrated previously to be copy number-dependent and independent of the site of integration (Dale, T., Krnacik, M. J., Schmidhauser, C., Yang, C. Q.-L., Bissell, M. J., and Rosen, J. M. (1992) Mol. Cell. Biol. 12, 905-914). The present study demonstrated that position-independent expression of the rat WAP -949/+2020 transgene was dependent on transgene spacing. Position-independent expression also was inhibited by an internal replacement of 49 base pair within the conserved GC-rich 3'-untranslated region (3'-UTR) with an identically sized nonspecific DNA sequence. Using electrophoretic mobility shift assays, nuclear factors isolated from mouse and human cells were shown to associate specifically with the rWAP 3'-UTR DNA, but not with the 3'-UTR containing the internal replacement or specific point mutations. Since a single copy of the 3'-UTR inserted 5' of the promoter could not rescue the 3'-UTR deletion, the 3'-UTR element does not appear to be functioning as either a classic enhancer or insulator element. However, the level of expression of rWAP transgenes was correlated with transgene association with the chromosomal scaffold in vivo.