HMGN1 (HMG-14) and HMGN2 (HMG-17) are nuclear proteins that bind specifically to nucleosomes, reduce the compactness of the chromatin fiber, and enhance transcription from chromatin templates. Here we report that many vertebrates contain an additional type of HMGN protein named HMGN3 (Trip 7). The human HMGN3 gene is located on chromosome 6 and spans 32 kilobase pairs, which is nearly 10-fold longer than the closely related HMGN2 gene. However, the intron/exon boundaries of the HMGN3 gene are identical to those of HMGN1 and HMGN2. Unique within the HMGN family, the HMGN3 transcript undergoes alternative splicing and generates two different variants, HMGN3a and HMGN3b. The shorter variant, HMGN3b, arises from an additional splice site that truncates exon V and causes a frameshift. The resulting HMGN3b protein lacks the majority of the C-terminal chromatin-unfolding domain. Both splice variants are found in many vertebrates from frogs to man and are expressed in many tissues. The pattern of tissue-specific expression differs considerably from those of HMGN1 and HMGN2 at both the mRNA and the protein level. Our results expand the multiplicity of the HMGN protein family and raise the possibility that these nucleosome-binding proteins function as co-activators in tissue-specific gene expression.