Both Sp-family factor Specificity protein 1 (Sp1) and LIM homeodomain transcription factor Lhx4 are involved in regulating the development of pituitary gland and nervous system in mammals. Sp1 gene mutation results in death of mouse embryo around day 11 of gestation, and mouse anterior pituitary development is severely hypoplastic after Lhx4 mutation. While Sp1 interacts with the related Lhx3 gene it is unclear whether Sp1 and Lhx4 also interact to regulate their physiological functions. The present study demonstrates that Lhx4 promoter is TATA-less and GC-rich and these sequences are conserved in different species. We have shown using site-directed mutagenesis and the Dual-Glo (TM) Luciferase Assay System that within the -515 to +36 bp basic activity regions of hLhx4 promoter the GC boxes were important for Sp1 regulation of the hLhx4 promoter. The electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments confirmed that Sp1 interacted with Lhx4 by directly binding to GC boxes located in Lhx4 promoter. We conclude Spl directly regulates Lhx4 gene expression. (C) 2007 Elsevier Inc. All rights reserved.