Acetylated polyamines, N 1 - and N 8 -acetylspermidine, are key intermediates in polyamine metabolism. Hexamethylenebisacetamide (HMBA) is a synthetic polyamine derivative known to induce in vitro biochemical and morphological differentiation of murine and human tumor cell lines. Deacetylation of HMBA and/or its metabolite N-acetyl-1,6-diaminohexane (NADAH) has been suggested to be catalyzed by N 8 -acetylspermidine deacetylase, a cytoplasmic enzyme involved in polyamine metabolism. The present investigation is focused on (1) the effect of N 1 - and N 8 -acetylspermidines, HMBA and NADAH on differentiation in Cloudman S91 melanoma cells, and (2) the effect of 7-[N-(3-aminopropyl)amino]heptan-2-one (APAH), a N 8 -acetylspermidine deacetylase inhibitor, on the effects of N 8 -acetylspermidine, HMBA, and NADAH on differentiation in Cloudman S91 melanoma cells. The biochemical signs of differentiation were measured as melanin content (absorbance at 475 nm) and tyrosinase activity (release of 3 H 2 O from 3 H-tyrosine) per μg protein. α-Melanocyte stimulating hormone (α-MSH) was used as a positive control. After 4 days of treatment, MSH at 100 nM raised the melanin to 293% of control (p < 0.05) and tyrosinase activity to 550% of control (p < 0.05). Spermidine did not produce any significant effect on these parameters in S91 cells. N 1 -acetylspermidine increased tyrosinase activity only at higher concentrations (100 and 1000 μM). However, 24-hour treatment of N 8 -acetylspermidine produced a dose dependent differentiation of S91 cells. At 10 −4 M, N 8 -acetylspermidine increased melanin by 85% and tyrosinase activity by more than 150% of control (p < 0.05). APAH alone had moderate effect on differentiation but potentiated and prolonged the effects produced by N 8 -acetylspermidine. HMBA decreased both melanin and tyrosinase activity in a dose dependent manner. After 4 days of treatment, 3 mM HMBA reduced melanin to 60% of control (p < 0.05) and tyrosinase activity to 4% of control (p < 0.05). NADAH is more active than HMBA. APAH was able to reduce the effects of HMBA (but not those of NADAH) on the parameters of differentiation. The results from the present study suggest that N 8 -acetylspermidine and HMBA have opposite effects on differentiation of S91 cells. The inability of APAH to counter NADAH-mediated decrease in tyrosinase activity suggests that APAH may be blocking the deacetylation of NADAH to an inactive metabolite.