A monospecific, polyclonal antibody to neutral collagenase purified from rat myometrial cells was used to examine decalcified sections of normal and rachitic rat long bones, including epiphyses, for presence of immunoreactive collagenase in situ. Reactive antigen was uniformly present in hypertrophic chondrocytes of articular and epiphyseal plate cartilage. Preincubation of antibody with excess homogeneously purified antigen blocked the staining, indicating specificity of the staining for the antigen. Reaction was present in the borders of the enlarging lacunae, suggesting functional importance of this enzyme in the process of lacunar enlargement prior to mineralization. No detectable enzyme was observed in osteoblasts, osteocytes, or osteoclasts. No difference between expression of collagenase by bone cells in rachitic or normal bone was seen. Thus, at the level of sensitivity of this procedure, neutral collagenase appears to function in mature rat bone only in enlarging chondrocyte lacunae.