Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) was used to analyse [S-35]-methionine-labelled proteins secreted in vitro by Sertoli cells when cultured in the presence or absence of enriched preparations of pachytene spermatocytes (SPC), early spermatids (SPT) or residual bodies/cytoplasts from elongated spermatids (RB/CES). The presence of germ cells modified the pattern of Sertoli cell secreted proteins in co-culture. Out of 21 Sertoli cell secreted polypeptide families visualized by 2D PAGE, one (referred to as number 12) was stimulated, whereas the secretion of polypeptides 1 and 3 was inhibited by all of the germ cell populations tested. Early spermatids and RB/CES both enhanced the secretion of protein number 10 and inhibited the production of protein 11. The RB/CES fraction specifically inhibited secretion of polypeptide 13. Of particular note was the finding that co-culture with early spermatids or RB/CES induced the secretion of a novel polypeptide, termed GIP (germ cell-induced protein), with an apparent molecular weight of 72 kDa and an isoelectric point of 5.9. Under the present experimental conditions, media conditioned by the different germ cell fractions inhibited the secretion of polypeptide 2 but enhanced the secretion of polypeptides 10 and 18; of note also was the finding that media conditioned by early spermatids or RB/CES induced the appearance of GIP. This study confirms and extends the concept that germ cells influence Sertoli cell function and that the effects observed differ according to the stage of development of the germ cells. However, the sensitivity of the 2D gel electrophoresis technique, and to some extent its reproducibility, limit its use for studying the paracrine control of Sertoli cells in culture.