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Abstract-The aim of this study was to estimate the intramuscular fat content (IMF) of porcine longissimus muscle using a handheld ultrasound device with a centre frequency of 3.2 MHz. The IMF estimation method from acoustic parameters obtained by the spectral analysis of ultrasonic echo signal is described. System specific effects and sound propagation effects on the spectral analysis were analyzed and corrected. RF data acquisition was performed at a commercial abattoir at 54 warm carcasses approximately 45 min post-mortem. Muscle specific acoustic parameters, i.e. attenuation, spectral slope, midband fit, apparent integrated backscatter, and cepstral parameters were extracted from the measured rf echoes.The cepstral parameters, i.e. integrated cepstrum and cepstral first peak value were the strongest indicators of IMF content changes (R 2 = 0.36). By multivariate regression analysis 66 % of the IMF variation (range 0.63 to 3.16 %) could be predicted with the proposed method (RMSEP = 0.35 %). The results suggest that this method is directly adaptable for utilization in a commercial abattoir for a non-invasive IMF estimation.