Factors influencing the survival of chlamydiae after freezing were reexamined. From the data presented, it is suggested that preservation of laboratory-grown chlamydiae is best achieved through the use of sucrose as the cryoprotective agent, in the presence of 10% serum. Dimethyl sulfoxide and glycerol are more toxic. The period of exposure to sucrose before freezing must be kept as short as possible and be at 4 degrees C rather than at room temperature. The rate of cooling during freezing in sucrose is not important; however, cooling at a rate slower than 1 degrees C/min should be avoided. Since chlamydial survival is increased by rapid thawing, the volume of the sample should be kept to a minimum. Thawed suspensions should be inoculated onto cell monolayers without delay. The application of these methods may increase the proportion of stored clinical specimens in which chlamydiae can be found.