Gibberellin A1 (GA1), 3-epi-GA1 GA17, GA19, GA20, and GA77 were identified by Kovats retention indices and full-scan mass spectra from gas chromatography-mass spectrometry analysis of a purified extract of mature seeds of photoblastic lettuce (Lactuca sativa L. cv. Grand Rapids). Non-13-hydroxylated GAs such as GA4 and GA9 were not detected even by highly sensitive radioimmunoassay. These results show that the major biosynthetic pathway of GAs in lettuce seeds is the early-13-hydroxylation pathway leading to GA1, which is suggested to be physiologically active in lettuce seed germination. Quantification of endogenous GAs in the lettuce seeds by gas chromatography-selected ion monitoring using deuterated GAs as internal standards indicated that the endogenous level of GA1 increased to a level about three times that of dark control 6 h after a brief red light irradiation, and that far-red light given after red light suppressed the effect of red light. The contents of GA20 and GA19 were not affected by the red light irradiation. Evidence is also presented that 3-epi-GA1 is a native GA in the lettuce seeds.