Two polypeptides with molecular masses of 34 and 30 kDa were copurified from rat liver during DNA affinity purification of a sequence-specific transcription factor binding to the footprint II sequence within the P2 promoter of the rat alpha1B adrenergic receptor (alpha1B AR) gene, and were identified by microsequencing their endoproteinase Lys-C-derived peptides as histone H1d and histone H1c, respectively. Histone H1 was previously reported to bind to the nuclear factor 1 (NF1) recognition sequence, although the specificity of this binding has been controversial. Here, DNA mobility shift and supershift assays, DNase I footprinting and mutational analyses indicated that the binding of histone H1 to the NF1 sites located within footprint II of the alpha1B AR gene P2 promoter is nonspecific. Transient cotransfections into Hep3B cells of histone H1d cDNA with CAT constructs containing promoter regions of different genes resulted in generalized and non-specific suppression of CAT activity. The histone H1d-mediated repression of the activities of the alpha1B AR gene P2/CAT or beta2 AR gene P(-186/1307)/CAT constructs was reversed by the cotransfection of a cDNA encoding the sequence-specific transcription factor NF1/X, and the fold increase in CAT activities was similar to that obtained in the absence of histone H1d. These results suggest that sequence-specific transcription factors counteract the histone H1-mediated transcriptional repression in vivo by a true activation, which is different from the in vitro antirepression in histone H1-repressed chromatin templates (Laybourn and Kadonaga, (1991) Science 254: 238-245).