The expression of transgene loci in mammals often occurs in a heterocellular fashion resulting in variegated patterns of expression. We have examined the effect of chromosomal integration site, copy number, and transcriptionally activating sequences on the variegation of a keratin 5-lacZ (K5Z) construct in the stratified epithelia of transgenic mice. lacZ expression in these mice is always mosaic, and the beta-gal activity per cell is usually higher in the lines with a higher proportion of expressing cells. Similar constructs, in which cDNAs were exchanged by lacZ sequences, showed no variegation. Also, when a strongly active, nonvariegating construct was coinjected with K5Z, most transgenic lines showed an almost homogeneous lacZ expression. The comparison of transgene arrays of different copies inserted at the same locus (obtained by using a lox/Cre system) showed that the reduction of copy number does not lead to an increase in the proportion of cells that express the transgene. Finally, in most of the variegating or nonexpressing lines the transgenes were located both at intermediate positions and at peritelomeric regions in the long chromosome arms. These findings suggest that the probability and efficiency of expression of K5Z genes depend on both long range chromosomal influences and on sequences in the transgene array.