Round spermatid energy metabolism is closely dependent on the presence of L(+)-lactate in the external medium. This L(+)-lactate has been proposed to be supplied by Sertoli cells in the seminiferous tubules. L(+)-lactate, in conjunction with glucose, modulates intracellular [Ca²⁺] in round spermatids and pachytene spermatocytes. In spite of this central role of L(+)-lactate in spermatogenic cell physiology, the mechanism of L(+)-lactate transport, as well as possible differentiation during spermatogenesis, has not been studied in these cells. Measuring radioactive L(+)-lactate transport and pHi changes with intracellular pH fluorescent probes, we show that these cells transport L(+)-lactate using monocarboxylate/H⁺ transport systems (MCT). RT-PCR, in situ mRNA hybridization, and immune cyto- and histochemistry data show that pachytene spermatocytes express mainly MCT1 and MCT4 isoforms of the transporter (intermediate and low affinity transporters, respectively) while round spermatids, besides MCT1 and MCT4, also show expression of the MCT2 isoform (high affinity). These molecular data are consistent with the kinetic data of L(+)-lactate transport in these cells that demonstrate at least two transport components for L(+)-lactate. These separate transport components reflect the ability of these cells to switch between the generation of glycolytic L(+)-lactate in the presence of external glucose, or the utilization of L(+)-lactate when this substrate is available in the external environment. The supply of these substrates would be regulated by the hormonal control of the Sertoli cell glycolytic activity.