Nucleus pulposus from the porcine intervertebral disc was separated chromatographically to discover substrates of microbial transglutaminase. Highly purified proteins were prepared, among them type II collagen, the major protein of the nucleus pulposus. Determination of substrates was performed by transglutaminase-mediated incorporation of biotinylated probes displaying several glutamine and lysine donor proteins. Type H collagen was only labeled if smaller nucleus pulposus proteins were present. One of the modulating proteins was serotransferrin, a lysine donor substrate of bacterial transglutaminase. An additional substrate was the carboxy-terminal propeptide of type II collagen, chondrocalcin. Chondrocalcin, a regulator of type II collagen fibrillogenesis, occurs abundantly in juvenile cartilage and nucleus pulposus. Accordingly, the protein may be regarded as an excellent additive for the preparation of injectable stem cells in nucleus pulposus-like matrices cross-linked by microbial transglutaminase.