A highly conserved region in the PTH promoter was identified using the basic local alignment search tool (BLAST) 2 Sequences comparison. Strong specific complexes were observed with a DNA probe that contained much of the computer-derived conserved sequence in the EMSA using bovine parathyroid gland (bPTG) nuclear extracts. Ethylation interference footprinting indicated that the major complex made contacts to a sequence strikingly similar to an Sp1 binding site. Sp3 was evident in the major DNA-binding complexes, whereas the contribution by Sp1 was substantially weaker. Specific binding by additional unidentified bPTG nuclear factors was also evident. Immunocytochemical and Western blotting analyses established that Sp1 and Sp3 were positively localized in the nuclei of chief cells of the bPTG and of the expected molecular weights, with particularly robust expression of Sp3. Affinity DNA-binding experiments using the bovine PTH Sp1 element demonstrated specific recovery of intact Sp3 and Sp1 proteins, although a significant portion of both proteins failed to interact with the affinity-tagged DNA. Treatment of the bPTG nuclear extracts with phosphatase, however, significantly increased the DNA-binding capacity of the Sp1/Sp3 complexes. Finally, transient transfection analysis indicated that the bovine Sp1-like element acted as an enhancer of heterologous gene expression. The present study identified an Sp1 element in the promoter of the PTH gene that represents a complex DNA-binding site involving interactions primarily with Sp1/Sp3 proteins. The data, therefore, highlight the likely involvement of the Sp family in regulating PTH gene expression through interactions with an Sp1 DNA element in the hormone's promoter.