Previous work has biochemically identified lysosomes containing calcium and calbindin-D28K (CaBP) in chick intestine that are sensitive to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] status. In the present work, lysosomal accumulation of 45Ca was optimal after 30 min of absorption from in situ ligated duodenal loops. The areas under the curves, defined as lysosomal fractions in Percoll gradients, were calculated, and values after 10, 20, 30 and 40 min of transport were (+D/-D ratio) 0.90, 1.2, 1.88, and 1.78, respectively. Lysosomal CaBP also increased in parallel with the time of absorption and was not due to nonspecific adsorption. When lysosomsal 45Ca was determined 2.5, 5, 10, 15, and 43 h after administration of 1.3 nmol nmol 1,25-(OH)2D3 or vehicle, the area ratios were 1.02, 1.47, 3.10, 1.88, and 1.29, respectively. Analyses of serum 45Ca in the same birds yielded a closely parallel time course with 1,25-(OH)2D3-dependent intestinal calcium absorption; values were 108 .+-. 12% (.+-. SE), 164 .+-. 29%, 300 .+-. 35%, 340 .+-. 39%, and 169 .+-. 8% of vitamin D-deficient control values at 2.5, 5, 10, 15, and 43 h, respectively. Immunoreactive CaBP in lysosomal fractions did not change significantly between 5-43 h after administration of seco-steroid. A similar series of experiments was conducted with microsomal membranes containing putative endocytic vesicles, which are believed to deliver calcium to the lysosomes. The brush border origin of the vesicles was supported by the internalization of anti-CaBP immunoglobulin G after 3 min of absorption. Accumulation of 44Ca by endocytic vesicles was subsequently found to be maximal after 20 min of absorption (+D/-D = 1.48), declining again at 30 min (+D/-D = 1.16), while CaBP levels in the same fractions remained unchanged between 0-30 min of absorption. These data together with the lower 45Ca specific activity in pinocytic vesicles relative to lysosomes suggest that lysosomes fuse with several endocytic vesicles in the interval of 20-30 min of absorption. Time-course studies with endocytic vesicles indicated that 1,25-(OH)2D3 stimulated uptake more rapidly than transport; 2.5, 5, 10, 15, and 43 h after seco-steroid administration, +D/-D ratios were 1.32, 1.87, 2.05, 1.72, and 1.36, respectively. CaBP levels in the same vesicle fraction did not correlate well with relative 45Ca content. The temporal dissociation between 45Ca content in endocytic vesicles vs. lysosomal and serum content at 5 h indicates that a 1,25-(OH)2D3-sensitive step occurs 5-10 h after seco-steroid administration, which promotes the delivery of vesicular contents to lysosomes to the basal lateral membrane.