Single-minded 1 and 2 are unique members of the basic helix-loop-helix Per-Arnt-Sim family as they are transcriptional repressors. Here we report the identification and transcriptional characterization of mouse Sim2s, a splice variant of Sim2, which is missing the carboxyl Pro/Ala-rich repressive domain. Sim2s is expressed at high levels in kidney and skeletal muscle; however, the ratio of Sim2 to Sim2s mRNA differs between these tissues. Similar to full-length Sim2, Sim2s interacts with Arnt and to a lesser extent, Arnt2. The effects of Sim2s on transcriptional regulation through hypoxia, dioxin, and central midline response elements are different than that of full-length Sim2. Specifically, Sim2s exerts a less repressive effect on hypoxia-induced gene expression than full-length Sim2, but is just as effective as Sim2 at repressing TCDD-induced gene expression from a dioxin response element. Interestingly, Sim2s bind to and activates expression from a central midline response element-controlled reporter through an Arnt transactivation domain-dependent mechanism. The differences in expression pattern, protein interactions, and transcriptional activities between Sim2 and Sim2s may reflect differential roles each isoform plays during development or in tissue-specific effects on other protein-mediated pathways.