Streptococcus bovis JB1 utilized glucose preferentially to lactose and grew diauxically, but S. bovis 581AXY2 grew nondiauxically and used glucose preferentially only when the glucose concentration was very high (greater than 5 mM). As little as 0.1 mM glucose completely inhibited the lactose transport of JB1. The lactose transport system of 581AXY2 was at least tenfold less sensitive to glucose, and 1 mM glucose caused only a 50% inhibition of lactose transport. Both strains had phosphotransferase systems (PTSs) for glucose and lactose. The glucose PTSs were constitutive, but little lactose PTS activity was detected unless lactose was the energy source for growth. JB1 had approximately threefold more glucose PTS activity than 581AXY2 (1600 versus 600 nmol glucose (mg protein)(-1)(min)(-1). The glucose PTS of JB1 showed normal Michaelis Menten kinetics, and the affinity constant (K-s) was 0.12 mM. The glucose PTS of 581AXY2 was atypical, and the plot of velocity versus velocity/substrate was biphasic, The low capacity system had a K-s of 0.20 mM, but the K-s of the high capacity system was greater than 6 mM. On the basis of these results, diauxic growth is dependent on the affinity of glucose enzyme II and the velocity of glucose transport.