In long-shoot buds of lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm), cone-bud initiation and gender differentiation occur in a site-specific manner: female cone buds are normally restricted to the distal portion, whereas male cone buds are located in the proximal portion. Application of a paste containing two plant growth regulators gibberellins A<inf>4</inf> + A<inf>7</inf> (GA<inf>4/7</inf>) combined with thidiazuron (TDZ) to long-shoot buds prior to cone-bud gender determination altered endogenous phytohormone profiles and induced female cone-bud formation in the proximal portion of the long-shoot bud, where male cone buds normally occur. Induced cone clusters observed in the following spring were either entirely female or a mixture of both female and male cones. Endogenous phytohormones in the long-shoot bud tissues were quantified by the stable isotope dilution method using high performance liquid chromatography-electrospray ionization tandem mass spectrometry in multiple-reaction monitoring mode. Applied GA<inf>4/7</inf> + TDZ led to increased concentrations of endogenous zeatin-type cytokinins, that is, trans-zeatin riboside and dihydrozeatin riboside, whereas concentrations of abscisic acid (ABA) and its catabolite, ABA glucose ester, were decreased, all relative to control, in untreated long-shoot bud tissue. Concentrations of extractable GA<inf>4</inf> and GA<inf>7</inf> declined in long-shoot bud tissues over 4 weeks following treatment with exogenous GA<inf>4/7</inf>. This study demonstrates that high levels of endogenous zeatin-type cytokinins, together with reduced levels of ABA, both induced by applied GA<inf>4/7</inf> + TDZ, are positively associated with an increased female cone-bud formation in long-shoot buds.