The mammalian protein synthesizing system is highly organized in vivo, and its substrate, tRNA, is channeled throughout the translation process. However, the cellular components responsible for this organization are not known. To examine this question a series of studies was carried out using intact and permeabilized Chinese hamster ovary cells. We show that cold shock dramatically reduces the protein synthetic capacity of these cells by as much as 95%. The loss of activity can be reversed by a short recovery period under conditions that allow energy metabolism to occur; transcription and translation during the recovery period are not needed. While individual components of the translation apparatus are not inactivated by the cold shock, the supramolecular organization of the system appears to be altered and F-actin levels are found to decrease. Resumption of protein synthesis during the recovery period coincides closely with the restoration of F-actin to normal levels. Moreover, disruption of actin filaments, but not microtubules, also leads to a major reduction in translation. These data support the conclusion that the cellular microfilament network plays an important role in the structure and function of the translation system and that perturbations of this network can have profound effects on protein synthesis.