Abstract: To increase the number of metaphase cells cultured in vitro, two bovine fibroblast cell lines（YFF and CNF） were frozen at 4℃ for different length of time prior to colchicine treatment , and then chromosomal specimen were prepared. The percentage of metaphase cells was examined under conditions above. Using this method, the variation rate of karyotype of YFF and CNF subcultured up to passage 20 were also analyzed. It was found that the percentage of YFF and CNF metaphase cells in treatment group were significantly higher than that in control group（P<0.05）, and the number of YFF and CNF metaphase cells obtained in 20 h treatment group were increased more than 6 fold as many as that in control group（P<0.01）, 31.7﹪and 40.2﹪ vs 4.7﹪and 6.4﹪ ,respectively. These data suggest that the method of freezing at 4℃ could be used for increasing the number of metaphase cells in vitro conveniently, and analyzing the variation rate of karyotype of cultured cells efficiently.