Myocardial tagging technique such as spatial modulation of magnetization (SPAMM) allows the study of myocardial motion with high accuracy. However, the accuracy of the estimation of tag intersection can be affected by tagline spacing. The aim of this study was to investigate the relationship between tagline spacing of SPAMM image and tagging contrast-to-noise ratio (CNR) in in-vivo study. Two healthy volunteers were undergone electrocardiographically triggered MR imaging with SPAMM-based tagging pulse sequence at a 1.5T MR scanner. Horizontally modulated stripe patterns were imposed with a range from 3.6 to 9.6 mm of tagline spacing. Images of the left ventricle(LV) wall were acquired at the mid-ventricle level during cardiac cycle with FE-EPI (TR/TE = 5.8/2.2 msec, FA= 10o. Tagging CNR for each image was calculated with a software which developed in our group. During contraction, tagging CNR was more rapidly decreased in case of narrow tagline spacing than in case of wide tagline spacing. In the same heart phase, CNR was increased corresponding with tagline spacing. Especially, at the fully contracted heart phase, CNR was more rapidly increased than the other heart phases as a function of tagline spacing. The results indicated that the optimization of tagline spacing provides better tagging CNR in order to analyze the myocardial motion more accurately.