N’-Acyl and N'-nitroso pyridine alkaloids were quantified by GC in burley tobacco genotypes varying in accumulation of pyridine alkaloids. Leaves were sampled during field growth and air-curing. N’-Substituted alkaloid identities were confirmed by GC and GC-MS; N’-acetylanatabine was newly identified. Alkaloid derivatives in lamina were in the following order of decreasing content averaged over the sampling dates. Acylated compounds: formylnornicotine (FNN), n-octanoylnornicotine (ONN), n-hexanoylnornicotine (HNN), formylanatabine (FAT), acetylnomicotine (ANN), n-butanoylnornicotine (BNN), and acetylanatabine (AAT). Nitrosamines: nitrosoanatabine (NAT), nitrosonornicotine (NNN), and 4-(N-methyl-N-nitrosamino)-l-(3-pyridyl)-l-butanone (NNK). Generally higher levels of N’-substituted pyridine alkaloids were found in lamina of the KY 78379 line (high nornicotine and total alkaloids) and B21 cultivar (high nicotine and total alkaloids) compared to LAB21 (low total alkaloids). N’-Acyl alkaloids occurred in green as well as cured leaves; N’-nitroso alkaloids were mainly present during curing. Thus, acylated alkaloids, unlike nitroso alkaloids, are apparent products of plant metabolism during late growth stages. © 1989, American Chemical Society. All rights reserved.