We have investigated the physiological importance of enzymic alterations in the rat pituitary during lactotroph involution induced by cessation of lactation. Lactating rats that had been suckling their young for 3 days were divided into two groups. One group was permitted to suckle their pups for up to 2 more days; the young were removed from the other group. At successive times, homogenates were prepared from their anterior pituitaries. The postlactating animals showed a rapid fall in plasma PRL levels, and this was accompanied by a transient rise in pituitary PRL content maximal 12 h after pup removal, which returned to control levels 36 h later. The pituitary homogenates were assayed for organelle marker enzyme activities. Both groups showed no significant alteration in pituitary protein or DNA content. The lactating group showed no change in the specific activity (milliunits per mg protein) of the following enzymes (organelle shown between parentheses): N-acetyl-β- glucosaminidase, acid PRL protease, β-glucuronidase, acid phosphatase, and cathepsin C (lysosomes); neutral α-glucosidase, (endoplasmic reticulum); 5′-nucleotidase and alkaline phosphatase (plasma membrane); malate dehydrogenase (mitochondria); lactate dehydrogenase (cytosol). The postlactating animals showed a transient increase in lysosomal enzyme activities, with the exception of cathepsin C, between 12 and 36 h after pup removal. This was particularly striking for PRL protease, which showed a 16-fold rise at 24 h. There were no significant alterations in neutral α-glucosidase, but both 5′-nucleotidase and alkaline phosphatase showed increased activities between 12 and 36 h after pup removal. There was no consistent alteration in the activities of lactate and malate dehydrogenases. These data provide biochemical evidence for the role of lysosomes in the degradation of PRL in the lactotroph involution after cessation of lactation.