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We measured translational diffusion of proteins in the cytoplasm and plasma membrane of the Gram-positive bacterium L actococcus lactis and probed the effect of osmotic upshift. For cells in standard growth medium the diffusion coefficients for cytosolic proteins (27 and 582 kDa) and 12-transmembrane helix membrane proteins are similar to those in E scherichia coli. The translational diffusion of GFP in L . lactis drops by two orders of magnitude when the medium osmolality is increased by ∼ 1.9 Osm, and the decrease in mobility is partly reversed in the presence of osmoprotectants. We find a large spread in diffusion coefficients over the full population of cells but a smaller spread if only sister cells are compared. While in general the diffusion coefficients we measure under normal osmotic conditions in L . lactis are similar to those reported in E . coli, the decrease in translational diffusion upon osmotic challenge in L . lactis is smaller than in E . coli. An even more striking difference is that in L . lactis the GFP diffusion coefficient drops much more rapidly with volume than in E . coli. We discuss these findings in the light of differences in turgor, cell volume, crowding and cytoplasmic structure of Gram-positive and Gram-negative bacteria. [ABSTRACT FROM AUTHOR]