Replication-defective retroviral or lentiviral vectors have been used for the production of transgenic animals. Chicken primordial germ cells (PGCs) are the precursors for ova and spermatozoa. Here, we describe the production of transgenic chickens via a germline transmission system using PGCs infected with a replication-defective lentiviral vector. PGCs were sorted with a fluorescence-activated cell sorter based on the expression of stage-specific embryonic antigen-1 from 2.5-and 5.5-day embryos. PGCs from both stages of embryo were infected with a lentiviral vector at a similar efficiency in vitro. PGCs were then transferred into the bloodstream of 2.5-day recipient embryos. The efficiency with which the PGCs were delivered and settled in the gonads was lower for PGCs from 5.5-day embryos than those from 2.5-day embryos when a limited number of PGCs was transferred, while the difference was not obvious upon the transfer of increased number of cells. Using a high number of 5.5-day PGCs infected with a lentiviral vector, transgenic chimeras (G(0)) with an acceptable efficiency for germline transmission were obtained. G(0) female chickens produced transgenic progeny (G(1)) with higher efficiency compared to G(0) male chickens. In G(1) transgenic chickens obtained by this method, enhanced green fluorescent protein was effectively expressed under the control of the actin promoter. (C) 2009. The Society for Biotechnology, Japan. All rights reserved.