Cell types in addition to those previously described (Kruse et al. 1963. J. Nat. Cancer Inst. 31:109; Kruse and Miedema. 1965. J. Cell Biol. 27:273) were found to form multiple-layered cultures by perfusion-culture technique. Dense populations containing 43 x 10(6) embryonic rat muscle (NF-ER) cells, 23 x 10(6) diploid human tonsillar (NF-JAM) cells, 77 x 10(6) human pleural effusion isolate (RPMI 2650) cells, 35 x 10(6) embryonic diploid human lung (Flow 2000) cells, 21 x 10(6) bovine lung (FB4BM) cells, 108 x 10(6) bat lung (Tb1Lu) cells, and 81 x 10(6) SV-40 virus-transformed embryonic diploid human lung (WI-38VA13A) cells were obtained in 6-14 days from dilute inocula in T-60 or T-75 flasks; these were equivalent to about 4, 3, 3, 4, 2, 4, and eight monolayers, respectively. Perfusion of an NF-ER culture for 6 wk with medium plus 10% whole calf serum yielded a cell density equivalent to 12 monolayers (140 x 10(6) cells per T-75 flask). This culture exhibited random labeling of nuclei from bottom to top after pulsing for 90 min with thymidine-(3)H. Medium plus 0.1% serum maintained NF-JAM cultures at constant viable cell numbers with virtual absence of thymidine-(3)H labeling. Similar results were obtained with WI-38 cultures, but WI-38VA13A cells continued active DNA synthesis and mitosis in medium with 0.1% serum to form 16-20 layers of cells (191-239 x 10(6) cells per T-75 flask) in 27 days. WI-38VA13A cells ceased proliferation and became nonviable rapidly in serumless medium.