1. Butyrate, a bacteria fermentative product in the colonic lumen, has been shown to produce a wide variety of biological effects in human cancer cells in vitro. However, there are pharmacological drawbacks associated with the use of butyrate therapy and there are limited published data on the structure-activity relationship of butyrate analogues in colorectal cancer cells. Previously, we determined structure-activity relationship using HT-29 human colorectal cancer cells. However, it was viewed as important to explore similar relationships in another colorectal cancer cell line. 2. Therefore, in the present study, the in vitro structure-activity relationship of butyrate analogues was examined by investigating their effects on apoptosis, cell proliferation, histone deacetylase (HDAC) activity and lactate dehydrogenase (LDH) leakage as a measure of cell toxicity in HCT-116 human colorectal cancer cells. 3. Of the 32 analogues tested, only 4-benzoylbutyrate, 3-benzo-ylpropionate, 4-(4-nitrophenyl)butyrate and 3-(4-fluorobenzoyl)propionate exhibited comparable biological effects to butyrate. The common structural properties of the compounds of interest were to lack amino or hydroxyl substitutions at the 2-, 3- and/or 4-position of the aliphatic moiety of butyrate. 4. The present study reveals a dissociation between the induction of apoptosis, inhibition of cell proliferation, HDAC activity and LDH leakage. The results indicate differential responses of butyrate analogues in HT-29 and HCT-116 colorectal cancer cells.