ABSTRACT Toxin-antitoxin systems are ubiquitous in the prokaryotic world and widely distributed among chromosomes and mobile genetic elements. Several different toxin-antitoxin system types exist, but what they all have in common is that toxin activity is prevented by the cognate antitoxin. In type I toxin-antitoxin systems, toxin production is controlled by an RNA antitoxin and by structural features inherent to the toxin messenger RNA. Most type I toxins are small membrane proteins that display a variety of cellular effects. While originally discovered as modules that stabilize plasmids, chromosomal type I toxin-antitoxin systems may also stabilize prophages, or serve important functions upon certain stress conditions and contribute to population-wide survival strategies. Here, we will describe the intricate RNA-based regulation of type I toxin-antitoxin systems and discuss their potential biological functions.