Hydroxyapatite (HA) is one of the most important calcium phosphates for hard tissue replacement in human bodies. Recently, cation substituted HA has been a research focus in order to enhance HA bioactivity and to adapt various application requirements. The study was aimed at investigating the contribution of zinc (Zn) and magnesium (Mg) ions when substituted into the structure of HA. The substituted samples were synthesized through a sol-gel route and then calcined at 800°C, 1000°C and 1200°C. Comprehensive characterization techniques, including transmission electron microscopy, BET surface area, X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetric and in-vitro analysis provided experimental evidence of the effects of ion substitution on the morphology, crystallite size, BET surface area, phase transformations, crystallinity, functional groups, thermal stability and bioactive behavior of HA. The results showed that as-synthesized zinc substituted hydroxyapatite (ZnHA) and magnesium substituted hydroxyapatite (MgHA) nanopowders consisted of flake-like agglomerates and the length of particles varied in the range 26-36 nm and 15-19 nm, respectively. The thermal stability and surface area was higher for ZnHA than MgHA nanopowder. In-vitro analysis of nanopowders on immersion in simulated body fluid (SBF) showed trend of alternate decrease and increase in pH of SBF confirming the bioactive behavior of nanopowders.