The putative citrate permease (Cit-P) plasmid DNA (pN7CP) from Lactococcus lactis subsp. lactis biovar diacetylactis (L. diacetylactis) NIAI N-7 was cloned. Cit-P+ transformants possessed pIL253 with a 3.8-kb fragment (pKAK1) or a 2.0-kb fragment (pKAK7). No XbaI site was present in the 3.8-kb of Xbal-Xbal fragment from pN7CP. It may thus be possible to obtain the 2.0-kb fragment by deleting the 3.8-kb fragment. Assessment was made of citrate-transporting activity in transformants KAK1 and KAK7. Several Cit(-) lactococci were transformed by pKAK7. Although all their transformants except KK-7LM and KK-7SL of Lactococcus lactis subsp. lactis (L. lactis) LM0230 and NIAI SLN were incapable of transporting citrate, transformants KK-7LM and KK-7SL showed the Cit(+) phenotype and citrate-transporting activity. L. diacetylactis and L. lactis were classified under citrate metabolism. However, the present findings strongly indicate some strains of L. lactis to quite likely be the original citrate utilizers but simply to have lost the Cit-P gene.