Lactoferrin expresses in a variety of tissues and involves in various aspects of host defense mechanisms. The lactoferrin gene is differentially regulated through multiple signaling pathways. Recently, an alternative form of human lactoferrin mRNA (Delta LF) was found in normal human tissues but absent from the tumor cells. In this study, we identified the transcription start sites of the Delta LF in mammary gland and bone marrow and demonstrated that the Delta LF is the product of an alternative (P2) promoter present in the first intron of the lactoferrin gene. The P2 promoter has high activity in Jurkat and U937 and low activity in RL95-2 and HEC-1B cell lines. Nonetheless, the promoter activity in HEC-1B cells was dramatically enhanced with overexpression of the Ets-1 transcription factor. The GFP-tagged lactoferrin is present in the cytoplasm whereas GFP-tagged Delta LF is found in both nucleus and cytoplasm as examined by fluorescence and confocal microscopy.