High-mobility group (HMG) proteins are architectural factors that have been shown to play a role in the transcriptional regulation of various mammalian genes. One family of HMG proteins, HMG-I/Y, is known to facilitate the initiation of gene transcription by modifying the conformation of DNA and recruiting transcription factors into an organized complex on transcriptional regulatory regions of specific genes. In many circumstances, the nuclear factor-kappaB family of transcription factors is involved in gene regulation that is mediated by HMG-I/Y. We will review the mechanisms by which HMG-I/Y proteins regulate gene transcription, give an overview of selected genes regulated by HMG-I/Y, summarize the potential roles of these genes in critical illnesses, and provide more detailed information about the role of HMG-I/Y in the regulation of nitric oxide synthase-2 during an inflammatory response, such as endotoxemia/sepsis.