In vivo calcium absorption was studied in normal and rachitic chicks. Cytochalasin B (CB) at a concentration of 25 microgram/ml added to the medium inside the duodenal lumen inhibited calcium absorption (20 min) from 82.5 +/- 1.9% of calcium absorbed in the controls to 59.2 +/- 3% in normal and from 70.0 +/- 2.3% to 47.0 +/- 2.1% in rachitic chicks. In vitro studies by everted ileal sacs of young rabbits also showed an inhibition of active transport of calcium due to CB. Whereas in the controls the ratio of 45Ca concentrations in serosal and mucosal media (60 min) was 7.2 +/- 0.32, the ratios were 5.24 +/- 0.52; 4.40 +/- 0.36; 3.40 +/- 0.42; 5.77 +/- 0.52; 1.38 +/- 0.08; and 1.06 +/- 0.02 in the presence of CB at concentrations of 5, 10 and 25 microgram/ml; colchicine 10(-4)M, Na citrate 0.02M, and heat-devitalized conditions, respectively. 45Ca concentration in the mucosal scrapings was also affected. It showed an increase from controls (15,101 +/- 404 cpm/mg) and correlated with CB concentration: 17,378 +/- 489, 19,015 +/- 1000, and 20,201 +/- 362 at 5, 10, and 25 microgram/ml, respectively. Dihydrocytochalasin B also inhibited active calcium transport and caused an increase in 45Ca concentration in the mucosal scrapings. Correlated electron microscopic studies showed certain changes in the brush border, especially in some actin microfilaments in the terminal web region. It seems that these morphological alterations may be related to transcytoplasmic movement of calcium.