A two step method to obtain mesenchymal free 3.5 day old chick embryonic notochords in vitro is presented. 1.) Notochords are isolated by mechanical microdissection from the embryos below the head and above the leg-buds. 2.) The dissected notochords are trypsinized to eliminate contaminating mesenchymal cells, while the perinotochordal sheath (PNS) is retained. After isolation and trypsinization, notochords are cut in standard 8mm lengths, explanted in vitro and incubated at 37°C. Immediately before incubation and after 3 and 6 days in vitro, notochords are fixed and stained to follow the morphological changes. The total DNA content of notochords is measured before and during maintenance in vitro to evaluate their metabolic activities. Results show that during the in vitro period, the isolated mesenchymal free notochordal fragments can conserve their characteristic architecture. The total DNA content measurements indicate proliferative activity and a high viability of the notochords in our in vitro system. In the present study, an isolation and in vitro method is offered which might be an effective tool to study the metabolic activities of chick embryonic notochords in vitro in comparison to in vivo behaviour, in order to study the underlying mechanism of notochord regression. © 1996 Kluwer Academic Publishers.