In living cells, the biochemical processes such as energy provision, molecule synthesis, gene expression, and cell division take place in a confined space where the internal chemical and physical conditions are different from those in dilute solutions. The concentrations of specific molecules and the specific reactions and interactions vary for different types of cells, but a number of factors are universal and kept within limits, which we refer to as physicochemical homeostasis. For instance, the internal pH of many cell types is kept within the range of 7.0 to 7.5, the fraction of macromolecules occupies 15%-20% of the cell volume (also known as macromolecular crowding) and the ionic strength is kept within limits to prevent salting-in or salting-out effects. In this article we summarize the generic physicochemical properties of the cytoplasm of bacteria, how they are connected to the energy status of the cell, and how they affect biological processes (Fig. 1). We describe how the internal pH and proton motive force are regulated, how the internal ionic strength is kept within limits, what the impact of macromolecular crowding is on the function of enzymes and the interaction between molecules, how cells regulate their volume (and turgor), and how the cytoplasm is structured. Physicochemical homeostasis is best understood in Escherichia coli, but pioneering studies have also been performed in lactic acid bacteria.