Polyamines are essential for optimal cell growth. The regulation of polyamine biosynthetic, but not catabolic, enzymes has been studied in detail. Because intracellular polyamine contents depend on both synthesis and catabolism, we studied the regulation of spermidine/spermine N1-acetyltransferase (N1SSAT), the first enzyme in polyamine catabolism. Steady-state RNA levels of N1SSAT increased 3-5 fold as human colon tumor-derived HCT116 cells traversed the log phase and entered the plateau phase. Depletion of cellular polyamines, using alpha-difluoromethylornithine, caused a decrease in the steady-state levels of both the 1.3-kb N1SSAT transcript and its 3.5-kb precursor, without affecting the stability of either RNA. N1SSAT enzyme activity was low in cells with normal polyamine contents but could be induced by heat shock. The level of induction of N1SSAT enzyme activity by heat shock on different days of growth correlated with N1SSAT RNA levels prior to heat shock and occurred without changes in levels of message after heat shock. Although non-heat-shocked cells containing normal polyamine contents expressed N1SSAT RNA but not enzyme activity, exogenous spermidine restored both RNA levels and enzyme activity in polyamine-depleted cells. This result suggests that the expression of N1SSAT enzyme activity, but not RNA, requires a change in the intracellular compartmentalization of spermidine. These data demonstrate that N1SSAT is regulated at both the transcriptional and posttranscriptional levels by conditions that arrest growth in HCT116 cells, and that both of these mechanisms are affected by endogenous polyamine contents.