BALB/3T3 fibroblasts (3T3) were observed to secrete latent, pepsin-activatable forms of cathepsin B and cathepsin L as well as an active form of beta-glucuronidase when cultured in the absence of serum. The secretion of these proteins was stimulated by the cation ionophore monensin: cathepsin B, 4.3-fold; cathepsin L, 7.2-fold; and beta-glucuronidase, 3.1-fold. These increases were accompanied by a 50% decline in cellular levels of the active forms of these enzymes and by the cellular accumulation of latent forms of cathepsin B and cathepsin L. Latent forms of beta-glucuronidase were not detected. In contrast, Moloney murine sarcoma virus-transformed BALB/3T3 fibroblasts (MMSV) secreted greatly increased amounts of latent cathepsin B (17-fold) and latent cathepsin L (27-fold), and moderately increased amounts of active beta-glucuronidase (2-fold) in a manner which was not further increased by monensin. The increased monensin-insensitive secretion of these lysosomal enzymes by MMSV cells may be due to a transformation-induced decrease in mannose 6-phosphate receptors. Thus, 3T3 cells bound the neoglycoconjugate pentamannosyl 6-phosphate-bovine serum albumin at 4 degrees C in a pentamannosyl 6 phosphate and mannose 6-phosphate-inhibitable manner, whereas MMSV cells showed no measurable cell surface mannose 6-phosphate receptor binding activity.