Mouse blood imaged using high-frequency ultrasound (US) is more echogenic in embryos than in adults. Studying changes in blood echogenicity in embryos may be of fundamental interest in studies on the genetic regulation of normal and abnormal blood development in mutant mice. Embryonic red blood cells (RBCs) are large and nucleated in midgestation but decrease in size and become enucleated as they mature. We therefore hypothesised that these structural alterations are responsible for variations in echogenicity of embryonic blood with gestational age and development. The objective of the current study was to quantify these structural changes in echogenicity (echo brightness) and apparent integrated backscatter (AIB) from embryonic blood at high US frequencies in vivo in mice. Results from anaesthetised pregnant mice studied using transcutaneous US showed that echogenicity of embryonic blood in the heart, aorta and umbilical cord and AIB within the heart chambers peaked at embryonic day (ED) 13.5 and then decreased progressively toward term. Between EDs 13.5 and 17.5 (near term), RBC mean cell volume decreased from 133 to 109 fL, haematocrit increased from 12 to 34%, and the percentage of nucleated RBCs decreased from 59 to 2%. Relative to younger ages, RBC nuclei at ED 13.5 were small and dense (pyknotic) which may have contributed to the peak in echogenicity and AIB at this age. To calculate the AIB, radiofrequency (RF) signals with centre frequencies of 28 MHz and 35 MHz were integrated over the 16- to 35-MHz and 21- to 42-MHz frequency range, respectively. At 28 MHz, mean apparent integrated backscatter of blood in the embryonic heart increased significantly from 0.0023 +/- 0.0004 Sr.cm(-1) (mean +/- SEM) at ED 12.5 to peak at 0.0037 +/- 0.0005 Sr.cm(-1) at ED 13.5. The mean AIB then decreased progressively with advancing gestation to 0.0002 +/- 0.0001 Sr.cm(-1) at ED 17.5. At 35 MHz, the mean AIB changed similarly with gestational age, except that values were lower than at 28 MHz at all ages. Higher attenuation of US at 35 MHz than at 28 MHz in tissue likely accounted for the lower AIB of blood insonified at 35 MHz. We speculate that developmental changes in red cell morphology are responsible for the observed changes in echogenicity and AIB of embryonic blood with gestational age in mice.