Compared with OFDM, Nyquist signal transmission has several unique advantages, such as lower receiver complexity, lower receiver bandwidths, and lower peak-to-average power ratios, which give better performances under fiber nonlinear impairments. Since the sinc-shaped waveforms in the time domain correspond to a rectangular spectrum in the frequency domain, the Nyquist pulses can also be obtained directly from the generation of a flat frequency comb with electro-optic modulators. In this paper, a complete theoretical analysis of Nyquist pulse generation with 3 comb lines generated with a single intensity modulator is developed. It is proved that both the amplitude and phase of the sidebands in the optical frequency comb are important for generation of Nyquist pulses. Theoretically, it is found that it is impossible to generate Nyquist pulses with more than 3 comb lines using a single intensity modulator even if 5 flat comb lines are realized. A Nyquist pulse can be generated with 9 comb lines by two cascaded intensity modulators, but the sum of the phase shift caused by the two DC voltages of the intensity modulators must be π.