Alkaloids and nitrite in tobacco leaves are known to be involved in the formation of tobacco-specific nitrosamines (TSNA). The formation is affected greatly by environmental conditions during curing and post-curing stages such as storage. To elucidate the correlation between alkaloid and nitrite contents and the TSNA formation more clearly, cured leaf samples of Burley tobacco with different concentrations of alkaloid and nitrite contents were treated using high-temperature conditions to promote TSNA formation. First, six cured leaf samples with different concentrations of alkaloid and nitrite contents were produced experimentally using a selected variety and modifying cultivation and curing practices. Secondly, heat treatment (HT) at 70 °C for 14 days was applied to them to maximize TSNA formation. The contents of alkaloids, nitrite and TSNA post-curing, and TSNA post-HT were determined using the JT standard method. As planned, samples of six kinds were obtained as combinations of three degrees of alkaloid contents (Low, < 10 mg/g; Middle, 10–50 mg/g; High, > 50 mg/g) and two degrees of nitrite content (Low, < 1.0 µg/g; High, > 1.0 µg/g). All six samples showed low TSNA contents of less than 1.0 µg/g immediately after curing. No positive correlation was found between TSNA contents and alkaloid or nitrite contents. After HT, TSNA contents increased extremely in samples with Middle and High levels of alkaloid content. The relation between the increased amounts of TSNA contents after HT and alkaloid and nitrite contents before HT was indicated. As described above, alkaloid and nitrite contents of cured leaves were related to increasing of TSNA contents depending on post-curing environmental conditions. To elucidate the TSNA formation mechanisms, further investigation is needed.