Fresh cytosols extracted from unfertilised eggs of Xenopus laevis contain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca2+ and is designated primary CSF (1°CSF). During storage of Ca2+-containing cytosols at 2°C, a stable CSF activity appears which is designated secondary CSF (2°CSF). In the present study, we report that 2°CSF activity can be induced in cytosols extracted from stage VI oocytes, unfertilised eggs, electrically activated eggs or blastulae, in the presence of Ca2+. Both the intensity and the rate of 2°CSF development are dependent on the concentration of Ca2+ ions added to the cytosol. At Ca2+ concentrations of 5–10 mM, 2°CSF activity reaches a maximum in about 7 days. Secondary CSF is relatively resistant to heat but loses all activity after 5 min at 70°C. When stored at −80°C, 2°CSF activity remains detectable for about 6 weeks. Cytological observations show that blastomeres arrested by microinjection of 2°CSF developed in cytosols of unfertilised eggs, activated eggs or blastulae contain metaphase chromosomes embedded in a bipolar spindle that has no asters developed at its poles. In contrast, blastomeres arrested by 2°CSF in cytosols of stage VI oocytes contain condensed chromosomes but no spindle is formed. The mechanisms of the development of 2°CSF in Ca2+-containing cytosols and its mode of action are discussed.