A novel series of aroyl-pyrrolyl-hydroxy-amides (APHAs) active as histone deacetylase (HDAC) inhibitors has been reported. The new derivatives were designed by replacing the benzene ring of the prototype 1 with both aromatic and aliphatic, monocyclic and polycyclic rings (compounds 3a-i), or by inserting a number of substituents on the methylene linker of 1 (compounds 4a-l). Compounds 3a-i and 4a-l were active at sub-micromolar level against the maize deacetylases HD1-B (class I), HD1-A (class II), and HD2. Tested at 5 microM against human HDAC1 and HDAC4, 3b, 4a, and 4j showed significant HDAC1 inhibition, whereas on HDAC4 only 4a was highly effective. On the human leukemia U937 cell line, the same compounds did not alter the cell cycle phases and failed in inducing apoptosis. However, they displayed granulocytic differentiation at 5 microM, with 3b being the most potent (76% CD11c positive cells). Tested to evaluate their effects on histone H3 and alpha-tubulin acetylation, 3b and 4a showed high H3 acetylation, whereas 4a and 4b were the most potent with alpha-tubulin as a substrate.