The partitioning of mouse/mouse hybridoma cell line BIF6A7, mouse/rat hybridoma PFU-83, and CHO DUKX B11-derived cell line BIC-2 in aqueous two-phase systems (ATPSs) of poly(ethylene glycol) (PEG) and dextran was studied. The partitioning of BIF6A7 was investigated systematically by using a statistical experimental design. The aims were to identify the key factors governing cell partitioning and to select ATPSs with suitable cell partitioning for extractive bioconversions with animal cells. The influence of five factors, i.e., the poly(ethylene glycol) molecular weight (PEG MW), dextran molecular weight (Dr MW), tie-line length (TLL), pH, and the ratio of potassium phosphate to potassium chloride, defined as the fraction KPi/(KPi + KCl), on BIF6A7 cell partitioning was characterized by using a full factorial experimental design. The cell partitioning ranged from complete partitioning into the interface to an almost complete partitioning to the lower phase. In all cases less than 1% of the cells partitioned to the top phase. The potassium phosphate fraction had the largest effect on cell partitioning. Low potassium phosphate fractions increased the proportion of cells in the lower phase. To a lesser extent the other factors also played a role in the cell partitioning. The best partitioning for the BIF6A7 cell line was obtained in ATPSs with PEG MW = 35 000, Dr MW = 40 000, TLL = 0.10 g/g, pH 7.4, and KPi/(KPi + KCl) = 0.1, where 93% of the cells were present in the lower phase. The previously reported partitioning of BIF6A7 cells in ATPS culture medium, corresponded well with the current findings. The partitioning of mouse/rat hybridoma cell line PFU-83 and CHO cell line BIC-2 was studied in an ATPS culture medium with PEG 35 000, dextran 40 000, TLL = 0.12 g/g, and hybridoma culture medium. Both cell lines partitioned almost completely into the lower phase. Moreover, the PFU-83 cell line was able to grow in the ATPS hybridoma culture medium. This ATPS hybridoma culture medium therefore seems to be suitable for extractive bioconversions with a wide range of hybridoma cell lines, provided that their product can be partitioned into the upper PEG-rich phase.