We have used isopycnic gradient ultracentrifugation to isolate a total lamellar body fraction (total-lb) from rat lung and then further subfractionated this using differential centrifugation to obtain two distinct subpopulations of organelles. When the total-lb was diluted to 0.25 M with sucrose and centrifuged at 8000 × for 30 min we obtained a fraction (IbA) that contained primarily intact classic-appearing Ib. When the supernatant was then centrifuged at 80,000 × g for 60 min we obtained a vesicular fraction (IbB). Whereas both fractions had an identical phospholipid composition, their enzyme profiles differed markedly. The IbA bad a higher level of βglycerophosphatase, while IbB had more 5' -nucleotidase. Moreover, IbB had a phospholipid:protein ratio of 9.2 while IbA had one of 6.3. An examination of the specific activity-time curves revealed that IbA had a curve that was broader and reached a peak earlier than IbB, but the downslopes of both curves were identical; they did not bear a classic precursor-product relationship to one another. The two fractions differed very significantly in their protein profiles. Whereas IbA contained a large amount of a 15-kDa protein with very small amounts of 35-, 37-, 38-, 45-, and 60-kDa proteins, IbB contained predominantly a 35-kDa protein with smaller amounts of 15-, 23-, 26-, 37-, 38-, 45-, and 60-kDa proteins. We suggest that IbB is surfactant taken back up into the alveolar type II cell, or a second release form of tissue surfactant, or a mixture of the two.