Trypsinized secondary cultures of various cell systems have been frequently used by different investigators as models to study, in vitro chemical transformation. In the present study, non-trypsinized primary cultures of fibroblasts derived from fetuses of 14 days gestational age have been used to find out the timings of in vitro chemical transformation by 20-methylcholanthrene and has been designated as PMM-14 cells. These PMM-14 cells have been compared with trypsinized cultures of the same cell systems. The criteria for neoplastic transformation considered in the study involved the appearance of morphological changes, indefinite growth in tissue culture, acquisition of tumorigenic potential in vitro as evidenced by in vivo tests and in vivo latency period for palpable tumor formation. The time span of neoplastic transformation of non-trypsinized embryo fibroblasts in culture was remarkably reduced in comparison with trypsinized cultures of same cell systems. Such discrepancy seems to be due to repeated use of trypsin which may replace many of vital cell surface molecules causing a delay in the expression of carcinogen-induced malignancy. Similarly, repeated subculture before transformation may also have a role in the delayed expression of malignancy.