Transgenic mice were developed which expressed human lactoferrin (hLF) in the mammary gland. This was achieved by using regulatory elements of bovine β-casein gene, rabbit β-globin intron II, and SV40 polyadenylation signal to control the expression of hLF cDNA. Twenty lines of transgenic mice were produced. Milk could be collected from four lines of transgenic mice. In two of them hLF was present in the milk at concentrations of 150~200 μg/ml. Mammary-specific hLF expression was not correlated to the transgene copy number. hLF RNA was only detected in the mammary gland of transgenic mice. The expressed RNA was correctly spliced at the exon/intron junctions. From these results, it may be concluded that the recombinant expression vector developed in this study is a good candidate to control high levels of foreign gene expression in the mammary gland of transgenic animals.