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Born in Rotterdam, after finishing secondary school in Dordrecht, Quispel studied classical philology from 1934 to 1941 at the Leiden University. At Leiden he also began to study theology, which he continued at the University of Groningen. Quispel completed his doctoral work in 1943 at Utrecht University with a dissertation examining the sources utilized in Tertullian's Adversus Marcionem. He devoted study to several Gnostic systems, particularly Valentinianism. In 1948-1949 he spent a year in Rome as a Bollingen fellow and was appointed Professor of the History of the Early Church at Utrecht University in 1951. Quispel served as a visiting professor at Harvard University in 1964-1965 and at the Katholieke Universiteit Leuven in 1968. He was engaged in first editing Nag Hammadi Codex I (the "Jung Codex") and devoted attention to the Nag Hammadi Library and particularly to the Gospel of Thomas throughout the rest of his career. Quispel also made contributions to the study of early "Jewish-Christian" traditions as well as Tatian's Diatessaron (a second-century gospel harmony). He died in El Gouna, Egypt.
- The Original Doctrine of Valentine, Amsterdam, North-Holland 1947.
- A Jewish Source of Minucius Felix, 1949.
- Gnosis als Weltreligion, 1951.
- Faust: Symbol of Western Man, 1967.
- Gnosis and The New Sayings of Jesus, Rhein-Verlag, 1971.
- The Birth of The Child: Some Gnostic and Jewish Aspects, Leiden: Brill 1973.
- From Mythos to Logos, Leiden: Brill 1973.
- Gnostic Studies, 2 vols., Istanbul, 1974.
- Tatian and the Gospel of Thomas: Studies in the History of the Western Diatessaron, Leiden: Brill 1975. ISBN 90-04-04316-0
- The Secret Book of Revelation: The Last Book of The Bible, New York: McGraw-Hill, 1979. ISBN 978-0070510807
- Gnosis and Psychology, Leiden: Brill 1973.
- Jewish and Gnostic Man, (Eranos Lecures) 1986.
- Gnostica, Judaica, Catholica. Collected Essays of Gilles Quispel, edited by Johannes van Oort, Leiden: Brill 2008.