A novel method for the determination of all eight kinds of variously 15N-enriched spermidines including the natural one is described using gas chromatography-mass spectrometry. After strict assignments of nitrogen(s) in fragment ions split from pentafluoropropionyl derivative of spermidine with 20 eV impact, six fragments (P7 and P10 containing N1 and N4 of spermidine, P8 and P11 containing N4 and N8, and P14 and P15 containing N1, N4, and N8 as designated in the text) were found to be useful for calculating relative contents of the various spermidines. The calculation was carried out by a computer program with the input of 20 relative intensities observed at m/z 259-261, 273-275, 379-381, 393-395, 436-439, and 464-467. Reliability of the method was examined by measuring known mixtures of authentic spermidines. In an experiment testing reproducibility, an average coefficient of variation was 4.8% with a reasonable accuracy for the eight spermidines. Linearity of relative ratios of 15N-enriched spermidines to the natural one was assured with an average correlation coefficient of 0.996. The present method was applied for spermidine synthase reaction, and it was confirmed that the enzyme from rat prostate could not discriminate against 15N of putrescine.