Thirteen cell lines derived from embryos of the mouse, rat and hamster were sampled at closely spaced intervals for cytology and spontaneous neoplastic transformation. Transformation, determined by growth of the cells as malignant neoplasms in compatible hosts, occurred in eight lines and appeared to be unrelated to time in culture, passage number, proliferative activity of the cells or type of serum used to supplement the culture medium. In an effort to establish cytologic criteria for malignancy of rodent fibroblasts, cultures were examined for 19 cytologic abnormalities. The percentage of cells or colonies showing each of the abnormal properties was recorded. Five of the 19 were found to correlate with neoplastic transformation in all cell lines. These were cytoplasmic basophilia, reduced cytoplasmic spreading on substrate, cording, high nuclear: cytoplasmic ratio and clumping. Each of these properties is amenable to quantitation or computerized image analysis. On the basis of the five cytologic criteria, the percentage of colonies diagnosed neoplastic, borderline and non-neoplastic was determined for each line. Although these percentages varied from one transplant generation to another, the percentage of neoplastic colonies tended to increase with time in all lines which became malignant, and the borderline colonies tended to decrease, suggesting a progression from borderline to neoplastic state.