The ternary complex factor Elk-1 belongs to the Ets oncoprotein family. We demonstrate that this transcription factor is localized predominantly in the nucleus, for which at least two regions of Elk-1 are required. One of these regions is part of the N-terminal ETS-domain, while the other encompasses amino acids 137-157. In conjunction with the ETS-domain, which mediates autonomous binding of Elk-1 to some DNA target sequences, the conserved B-region is both necessary and sufficient for ternary complex formation with the c-fos serum response element and the serum response factor. However, the B-region must be linked to the ETS-domain by a spacer. Furthermore, the B-region impedes autonomous DNA-binding, possibly by masking the ETS-domain. A point mutation within the ETS-domain, homologous to the ts1.1 point mutation of v-Ets in the E26 virus, affects DNA-binding of Elk-1 in a temperature-dependent manner, which by analogy might be causative for the altered phenotype of ts1.1 E26. Finally we show that amino acids 83-428 contribute to Elk-1 mediated transactivation. In particular, the region 376-404 is indispensable for transactivation, while flanking amino acids on both sides are only required for enhancement of transcriptional efficacy.