The draft genome of the diatom Thalassiosira pseudonana was searched for DNA sequences showing homology with long-chain acyl-coenzyme A synthetases (LACSs), since the corresponding enzyme may play a key role in the accumulation of health-beneficial polyunsaturated fatty acids (PUFAs) in triacylglycerol. Among the candidate genes identified, an open reading frame named TplacsA was found to be full length and constitutively expressed during cell cultivation. The predicted amino acid sequence of the corresponding protein, TpLACSA, exhibited typical features of acyl-coenzyme A (acyl-CoA) synthetases involved in the activation of long-chain fatty acids. Feeding experiments carried out in yeast (Saccharomyces cerevisiae) transformed with the algal gene showed that TpLACSA was able to activate a number of PUFAs, including eicosapentaenoic acid and docosahexaenoic acid (DHA). Determination of acyl-CoA synthetase activities by direct measurement of acyl-CoAs produced in the presence of different PUFA substrates showed that TpLACSA was most active toward DHA. Heterologous expression also revealed that TplacsA transformants were able to incorporate more DHA in triacylglycerols than the control yeast.