Cartilage is encountered in the skeletons of many advanced invertebrates, yet it never calcifies or is replaced by bone. In an attempt to account for the absence of bone in invertebrates, we tested a hypothesis proposing that absence or inadequate quantities of several enzymes associated with vertebrate osteogenesis may underlie the failure of the invertebrates to evolve bone. The enzymes examined were alkaline phosphatase, alanyl beta-naphthylamidase, and neutral protease. Their activities were measured in the gill cartilage of the Atlantic horseshoe crab, Limulus polyphemus, and the odontophore cartilage of the marine whelk, Busycon canaliculatum. Animals were collected from the Cape Cod area. Samples of cartilage of Limulus perichondrium, various non-skeletal tissues, and neonatal rat calvaria, the latter as a reference standard, were homogenized in 0.1 M phosphate buffer (pH 7.1) and analyzed for protein content and the above-mentioned enzyme activities. Alkaline phosphatase specific activity was readily detected in most tissues except the invertebrate cartilage specimens in which it was present only at near-trace levels. Naphthylamidase and protease activities were present in all tissues. In a single experiment, higher phosphatase values were recorded for Limulus cartilage retaining perichondrium, but in a subsequent trial assaying cartilage retaining perichondrium, denuded cartilage, and isolated perichondrium separately, it was demonstrated that phosphatase activity resided primarily within the perichondrium. Exposure of thick cryostat sections to p-nitrophenyl phosphate confirmed the suspicion that alkaline phosphatase activity was present principally in the perichondrium.(ABSTRACT TRUNCATED AT 250 WORDS)