The impact of transient gene expression approaches (TGE) on the rapid production of recombinant proteins is undisputed, despite that all efforts are currently relying on two host cell families only, namely HEK293 derivatives and CHO cell line(s). Yet, the increasing complexity of biological targets calls for more than two host cell types to meet the challenges of difficult-to-express proteins. For this reason, we evaluated the more recently established novel CAP-T® cell line derived from human amniocytes for its performance and potential in transient gene expression. Upon careful analyses and adaptation of all process parameters we show here that indeed the CAP-T® cells are extremely amenable to transient gene expression and recombinant protein production. Additionally, they possess inherent capabilities to express and secrete complex and difficult target molecules, thus adding an attractive alternative to the repertoire of existing host cell lines used in transient production processes. Biotechnol. Bioeng. 2012;109: 2250–2261. © 2012 Wiley Periodicals, Inc.