3-Hydroxypropionic acid (3-HP) is an important platform chemical from which several commodity and specialty chemicals can be generated. The present investigation focuses on the construction and evaluation of a recombinant strain Escherichia coli SH254 that produces 3-HP from glycerol. The strain was developed by cloning two genes, dhaB of Klebsiella pneumoniae DSM 2026 encoding glycerol dehydratase and aldH of E. coli K-12 MG1655 encoding aldehyde dehydrogenase, respectively. In vitro assays of crude enzyme extract of glycerol dehydratase (DhaB) showed 37.0 U mg(-1) protein on glycerol with coenzyme B(12), and partially purified aldehyde dehydrogenase (AldH) exhibited 22.8 U mg(-1) protein on 3-hydroxypropionaldehyde (3-HPA) with NAD(+) as a cofactor. When cultivated aerobically on a glycerol medium containing yeast extract, the recombinant E. coli SH254 produced 3-HP at a maximum of 6.5 mmol l(-1) (0.58 g l(-1)), The highest specific rate and yield of 3-HP production were estimated as 6.6 mmol g(-1) cdw h(-1) and 0.48 mol mol(-1) glycerol, respectively. Although not optimized extensively, this study is encouraging for further development of a bioprocess to produce 3-HP from glycerol. (c) 2008 Elsevier Ltd. All rights reserved.