We have investigated the changes in biochemical markers and in pyridinium cross-links in bone in hypophosphatemic rats. Six-week-old female Wistar rats were divided into two groups (normal diet and a phosphate-deficient diet) and fed for 8 weeks. A low phosphate intake caused a significant difference in the concentrations of osteocalcin and alkaline phosphatase with advancing rachitis as well as an increase in bone resorption marker concentrations in urine. Femur biochemical analysis revealed a significant (p < 0.005) increase in deoxypyridinoline per mole collagen in the phosphate-deficient group which suggested that urinary excretions of pyridinium cross-links might reflect not only bone resorption but also increased pyridinium cross-links in bone matrix collagen. Our results demonstrate that a low phosphate intake causes an increase of pyridinium cross-link formation as well as a discrepancy between the circulation levels of alkaline phosphatase and osteocalcin with advancing rachitis. These alterations induced by low phosphate intake should be considered when interpreting the values of biochemical markers.