Surfactant protein B (SP-B) is critical for normal lung function. Previously we demonstrated that prolonged treatment of human adenocarcinoma H441 cells with 12-O-tetradecanoyl phorbol-13 acetate (TPA), an activator of protein kinase C, represses surfactant protein B gene transcription. We localized the regulatory DNA elements responsible for this effect to -140/-65 bp of the SP-B gene proximal promoter, a region which contains two thyroid transcription factor (TTF-1) binding sites and a hepatocyte nuclear factor 3(HNF3) element. Using electrophoretic mobility shift assay (EMSA), no new proteins binding to the -140/-65 fragment were identified in nuclear extracts of TPA-treated H441 cells. However, binding activity of all the three bands observed with control nuclear extracts were reduced after TPA treatment (10 nM) of cells for 24 h. To examine individual transcriptional factors, we made double stranded (ds) oligonucleotides for factors binding within or just outside the - 140/-65 region. In a time course study, binding activity using ds HNF3 as a probe was reduced ≈ 50% in nuclear extracts prepared after 2 h of TPA treatment. Similar results were observed for HNF3 protein analyzed by Southwestern analysis. EMSA with ds TTF-1 oligonucleotide resulted in 2 retarded bands and binding for both was reduced after 8 or more h of TPA treatment. A single band for TTF-1 oligonucleotide was detected by Southwestern blot which decreased with prolonged TPA treatment similar to the EMSA results. These kinetics for loss of TTF-1 binding activity are consistent with those for down regulation of SP-B gene transcription (at ≥8 h). By contrast, binding activity of general nuclear factors (AP2, SP1 and TBP) present outside of this region were not significantly affected by TPA treatment. We conclude that down regulation of SP-B gene expression by TPA involves decreased binding of the transcription factors HNF3 and TTF-1. These factors may be directly or indirectly affected by agents that act through protein kinase C.