Vitamin D-deficient, second generation, rachitic rats showed significant decrease in bone Gla protein (BGP) levels in circulation and in the skeleton. 1,25 dehydroxyvitamin D3 (1,25(OH)2D3) exhibited the most potent influence on serum BGP levels in a dose-dependent manner. At a dose 25 ng/100 g body weight 1,25(OH)2D3 showed a cumulative effect, i.e., the longer the treatment, the more circulating BGP was detected. 24,25 dehydroxyvitamin D3 (24,25(OH)2D3) at the same doses did not show similar effect on the serum BGP levels, regardless of the serum calcium levels. Bone BGP levels assayed at various sites representing endochondral and intramembranous ossification demonstrated an opposite pattern. 1,25(OH)2D3 administration was not sufficient to restore bone BGP levels to normalcy, whereas in animals treated with 24,25(OH)2D3, bone BGP and calcium levels were significantly higher than control (Vitamin D3-repleted) levels. The present results can be explained by the dual action of 1,25(OH)2D3 on both synthesis and release of BGP by bone turnover, whereas 24,25(OH)2D3 stimulates synthesis and accumulation of BGP in bone. These observations imply that caution is required in the interpretation of clinical data based solely on serum BGP determination.