Lembehyne A (LB-A), a spongean polyacetylene, induced neuronal cell differentiation in a neuroblastoma cell line, Neuro 2A. The LB-A treatment of Neuro 2A cells predominantly resulted in a morphological change with bipolar neurites. The acetylcholinesterase activity of Neuro 2A was also increased by the treatment of LB-A. Furthermore, the cell cycle of Neuro, 2A cells was found to be specifically blocked at the G1 phase by LB-A. The structure-activity relationship study using the LB-A analogues revealed the importance of the terminal 1-yn-3-ol and unsaturated long-chain alkyl moieties for the neuronal differentiation activity of LB-A.