Alkanolamines used for CO2-capture in natural gas (NG) may be released to the marine environment from coastal and offshore NG facilities. In this study biodegradation of alkanolamines in seawater was investigated. Screening of 20 alkanolamine candidates for ultimate biodegradability showed that biodegradation rates followed the order primary = secondary > tertiary > sterically hindered alkanolamines. Biodegradation studies of monoethanolamine (MEA), diethanolamine (DEA) and methyl-diethanolamine (MDEA) at seawater temperatures of 10–32 °C were performed with respirometric and chemical analyses, showing that temperature-related biodegradation was more pronounced for DEA than for MEA, while MDEA remained recalcitrant at all temperatures. No effect of concentration was detected when DEA and MDEA biodegradation was compared at initial concentrations of 2 and 5 mg/l. PCR-DGGE analyses of bacterial 16S rRNA gene fragments from seawater samples collected during the biodegradation experiments at 20 °C revealed one major band associated with DEA biodegradation. Analyses of the 16S rRNA gene fragments by cloning and sequencing showed predominance of the class Alphaproteobacteria in all samples, but biodegradation of DEA at 20 °C resulted in an increased fraction of Gammaproteobacteria. Sequence analyses of cloned inserts were performed after suppressive subtraction hybridization (SSH) cDNA PCR amplification of genes expressed during growth of the strain Pseudomonas sp. P6 on the alkanolamine 1-aminopropan-2-ol. The results of these analyses indicated that gene expression was associated with cobalamine (vitamin B12) synthesis with 1-aminopropan-2-ol incorporation in the vitamin B12 complex, and with putative enzymes involved in the glyoxylate and tricarboxylic acid cycles. In addition, the results suggested a general up-regulation of transcriptional and translational activities, including amino acid metabolism.