The relative effect of five commonly used culture media (MEM, BME, McCoy's 5A, M199 and HMEM) on the average nucleolar number/cell, the average volume/nucleolus and the total nucleolar volume/cell was examined during in vitro senescence of WI-38 human fetal fibroblasts. Statistical analyses show that cells aging in MEM show a higher number of nucleoli/cell than that of cells aging in any other medium. For cells aging in the other four media, there are no significant differences in the average number of nucleoli/cell. Linear regression analysis shows that in all cases there is a linear decrease in the average number of nucleoli/cell as a function of PDL. Statistical analyses show that the average volume/nucleolus is significantly greater for cells aging in M199 than in any other medium. Cells aging in HMEM show smaller average nucleolar volume than cells aging in M199, but display larger volumes than that of cells aged in BME, McCoy's 5A, or MEM. Cells aging in BME and McCoy's 5A media show no significant difference among each other in terms of average nucleolar volume, but a difference in this parameter is noted in cells aging in BME and MEM. A linear regression analysis shows that the average volume/nucleolus increases linearly as a function of age for cells grown in all five media. Analysis of the total nucleolar volume/cell in the five media shows that cells aging in M199 and HMEM are not significantly different from each other in terms of this variable, but show significantly larger volumes than those of cells aging in BME, McCoy's 5A and MEM. Cells aging in BME, McCoy's 5A and MEM display no significant difference with regard to this parameter. Linear regression analysis shows a positive linear relationship between the PDL and the total nucleolar volume/cell. The relative effects of all five media are not the same on the three cellular variables studied during in vitro aging of WI-38 cells. We, therefore, suggest that one should note this medium differential in order to allow meaningful comparison of results on possible changes in various morphological parameters during in vitro senescence of diploid human fibroblasts.