Nitrogen metabolism was investigated under shoot‐forming (SF) and non‐shoot‐forming (NSF) conditions in cultured cotyledon explants of Pinus radiata by following the incorporation of [14C]‐l,2‐acetate into various metabolites. Early in culture, the lipid fraction contained the most 14C; however, this percentage decreased in favor of increased label in the amphoteric fraction. Label in the amphoteric fraction of SF cultures decreased by day 21 but plateaued in NSF cultures at this time. Radioactive labeling of the principle nitrogen metabolites, glutamate and glutamine, which made up the majority of the amphoteric fraction, paralleled labeling patterns in the amphoteric fraction. Percentage label in glutamate remained at similar levels throughout the 21‐day culture period for both SF and NSF cultures. Specific activity of glutamate (kBq mg‐1) was significantly greater during promeristemoid formation in SF compared to that in NSF tissues. Glutamine labeling increased during shoot bud initiation in SF cultures, but dropped to lower levels during shoot bud development. In contrast, in NSF cultures, there was a continual and substantial increase in glutamine labeling throughout the 21‐day culture period. These trends were similar when the specific activities of glutamine were determined, as there was a continual decrease from culture initiation to the end of shoot bud differentiation in SF cultures. In NSF cultures, in contrast, specific activity of glutamine increased substantially from day 5 to 21 relative to that in SF cultures. The nitrogen assimilation enzymes glutamate synthase and glutamine synthase increased in activity from day 0 to 21 for both SF and NSF tissues. Enzyme activities for glutamate dehydrogenase were similar in both treatments to day 10 in culture but subsequently diverged, with activities in NSF cultures being substantially greater than those of SF cultures by day 21. Taken together, labeling and enzyme data indicate that nitrogen metabolism is enhanced during culture, especially in SF tissues at the time of promeristemoid formation, and in non‐organ‐forming tissue senescence‐like metabolism was exhibited later in culture.