The gp330/Megalin/LRP-2 protein belongs to the low-density lipoprotein receptor gene family and is believed to function as an endocytic receptor for the uptake of lipoproteins and many other ligands. Other functions of this protein may include a role in calcium sensing in the parathyroid glands and other tissues. In order to study the transcriptional regulation of the human LRP-2 gene, a clone containing the 5'-flanking region was isolated from a genomic DNA library, and a transient transfection protocol for primary bovine parathyroid cells was established. RNA mapping techniques located the transcriptional start site 136 bp upstream of the initiation codon. Transient expression in several cell types, including primary parathyroid cells, and in vitro transcription in HeLa cell nuclear extracts showed that sequences between -120 and -35 were important for activated transcription. This region contains consensus binding sites (GC boxes) for transcription factor Sp1. Mutation of the GC boxes abolished binding of Sp1 in vitro and resulted in reduced transcription in vitro and in transfected cells. Furthermore, Sp1 stimulated transcription when tethered to the LRP-2 core promoter through a heterologous DNA-binding domain. Through site-directed mutagenesis, we identified a novel atypical TATA element with the sequence TAGAAAA. Intriguingly, this sequence motif was shown previously not to mediate transcription in a systematic mutational analysis of the TATA motif. Possible roles of this novel TATA element in the regulation of transcription initiation are discussed. The isolation and characterization of the LRP-2 promoter and the 5'-flanking region and the establishment of a transient expression assay in primary parathyroid cells will facilitate studies on the regulatory mechanisms of the LRP-2 gene and of other genes expressed in the parathyroid glands.