The importance of intragenic and 3' flanking sequences in the control of the temporal, hormonal and tissue-specific expression of milk whey acidic protein (WAP) has been demonstrated in transgenic mice. Mouse lines carrying a 4.3 kb genomic clone containing the entire rat WAP gene minus 200 bp of the first intron with 0.949 kb of 5' and 1.4 kb of 3' flanking DNA were generated. In eight of nine independent lines of mice analyzed, WAP transgene expression was detected at levels ranging from 1% to 95% (average, 27%) of the endogenous gene. The transgene was expressed preferentially in the mammary gland. Although developmentally regulated during pregnancy and lactation, the temporal pattern of WAP transgene expression differed from the endogenous gene. A precocious increase in expression of the transgene was detected at 7 days of pregnancy, several days earlier in pregnancy than the major increase observed in endogenous mouse WAP mRNA. The rat WAP transgene was translated and secreted into the milk of transgenic mice at levels comparable to the endogenous mouse WAP. This is the first report of a gene that is negatively regulated in dissociated cell cultures as well as in transfected cells, yet is expressed efficiently in the correct multicellular environment of the transgenic mouse.