Abstract Signal transduction systems in bacteria and archaea link environmental stimuli to specific adaptive cellular responses. They control gene expression, motility, biofilm formation, development and other processes that are vital to survival. The microbial signal transduction (MiST) database is an online resource that stores tens of thousands of genomes and allows users to explore their signal transduction profiles, analyze genomes in bulk using the database application programming interface (API) and make testable hypotheses about the functions of newly identified signaling systems. However, signal transduction in metagenomes remained completely unexplored. To lay the foundation for research in metagenomic signal transduction, we have prepared a new release of the MiST database, MiST 4.0, which features over 10 000 metagenome-assembled genomes (MAGs), a scaled representation of proteins and detailed BioSample information. In addition, several thousands of new genomes have been processed and stored in the database. A new interface has been developed that allows users to seamlessly switch between genomes and MAGs. MiST 4.0 is freely available at https://mistdb.com; metagenomes and MAGs can also be explored using the API available on the same page.