Electron tomography (ET) has been used to reconstruct in situ individual 50S ribosomal subunits in Escherichia coli rifampicin-treated cells. Rifampicin inhibits transcription initiation. As a result, rapid degradation of preformed mRNA and dissociation of 70S ribosomes give accumulation of free subunits. In the 50S subunit, the L1 stalk, the L7/L12 stalk, the central protuberance (CP), and the peptidyl transferase center (PTC) cleft are the most dynamic and flexible parts in the reconstructed structures with clear movements indicated. Different locations of the tunnel in the central cross-sections through the in situ 50S subunits indicate the flexible nature of the pathway inside the large ribosomal subunit. In addition, gross morphological heterogeneity was observed in the reconstructions. Our results demonstrate a considerable structural variability among individual 50S subunits in the intracellular environment.