To investigate the sensitivity of guinea pig tissues to ascorbic acid depletion, as distinct from inanition, young male guinea pigs were maintained with either ascorbic acid restriction or total diet restriction for 8 weeks. One group (A) received no ascorbic acid for 3 weeks, then 0.5 mg/d for 5 weeks; one group (B) was weight-matched by restricted food intake to the first group; a third (marginally deficient) group (C) received 1 mg ascorbic acid/d throughout; a fourth was weight-matched to this group (D); and a fifth group received the control diet ad lib. (E). Both of the groups with restricted ascorbic acid intakes (A and C) developed very low tissue ascorbic acid contents, but only the first group (severely deficient group A) also exhibited a severely reduced growth rate. This group also exhibited reduced femur calcium and hydroxyproline contents and reduced skin hydroxyproline content. These changes were not seen in the corresponding weight-matched group (B). Neither plasma alkaline phosphatase (EC 3.1.3.1) activity, nor a variety of indices of vitamin D status exhibited changes which could be attributed specifically to reduced ascorbic acid intake and hence to lowered tissue ascorbic acid content. It is concluded that low tissue ascorbic acid levels in guinea pigs clearly alter the connective tissue composition of growing femur and skin, but do not necessarily produce a major, specific effect on vitamin D status. Moreover, the control of inanition is crucial to permit interpretation of the changes in metabolism that are caused by ascorbic acid deficiency.