Because collagen is the major target in the enzymatic dissociation of the pancreas for islet isolation, we determined the amount of collagen and its distribution in a comparative study comprising normal pancreata of rat, dog, man, young pig, and adult pig. Collagen content was determined using a colorimetric method and its distribution was assessed in tissue sections stained with Sirius red. The collagen content is relatively low in the rat and adult pig pancreas, and the amount of collagen is relatively low in the septa of the rat and dog pancreas. Not the amount of collagen in the septa but collagen in the rest of the pancreas, mainly located between the acini, seems to determine the dissociation of the pancreatic tissue. This can be exemplified by the higher islet yields obtained from the adult vs. the young pig pancreas; the latter contains a higher total amount of collagen but a similar, relatively high, amount of collagen in the septa. A high amount of collagen surrounding the islets seems to be of secondary importance in islet isolations, because yields of the same magnitude are obtained from the canine and human pancreas containing a relatively low vs. high amount of collagen around the islets but a similar total collagen content. The rat pancreas contains both a low total amount of collagen and a high amount of collagen around the islets; therefore, the general experience that islet isolation procedures are effective in rats can be readily understood.