In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector (TM) technology. After starting with a heterogenous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofetion experiments, transfection with 5 mu g of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 ph (.) cells(-1 .) day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 ph (.) cells(-1) (.) day(-1) in serum-dependent cultivation, a significant decreased that resulted in range q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PHA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development.