In almost all cases, an audio file has no single frequency. A sound in which the sound wave has a single frequency, is (typically) a pure sine tone, and sounds like this:
http://www.wolframalpha.com/input/?i=sound+440+Hz&a=*MC.~-_*PlaySoundTone-&a=*FS-_**DopplerShift.fo-.*DopplerShift.vs-.*DopplerShift.c--&f3=10+m/s&f=DopplerShift.vs_10+m/s&f4=340.3+m/s&f=DopplerShift.c_340.3+m/s&a=*FVarOpt.1-_***DopplerShift.fo-.*DopplerShift.fs--.***DopplerShift.DopplerRatio---.*--&a=*FVarOpt.2-_**-.***DopplerShift.vo--.**DopplerShift.vw---.**DopplerShift.fo-.*DopplerShift.fs---
This is a pure 440 Hz sine wave. (It was not possible to make a proper link of this, due to MarkDown limitations.)
A general sound, such as a recording (of speech, music, or just urban noise), consists of (an infinite number of) combinations of such sine waves, superimposed. That is, if you were to draw the graph of pressure vs. time (at a given point in space) of the wave, or, (more or less) equivalently, the position of the speaker's membrane as a function of time, it would hence not be a pure sine wave, but something much more complicated. (Indeed, how could all the information of a Beethoven symphony be represented in a simple sine wave, that is completely determined by only its frequency, a single number?)
The sampling rate of a digital recording is merely the number of samples per second of the sound wave. Indeed, a physical sound wave has an amplitude p(t) at each time, so, because there are an infinite number of times t between 0 s and 10 s (say), theoretically, to save the audio we would need an infinite number of bytes (each sample requireing a fixed number of bytes -- for instance, a 16-bit recording utilizes 16 bits, or 2 bytes, per sample -- of course, the higher the "bit number" is, the higher quality we get; for a 16-bit sound, we have 216 = 65536 levels to choose from when specifying a single sample). In practice, a sound is sampled, so that the amplitude p(t) is saved only at fixed intervals. For instance, a typical audio CD has a sampling rate of 44.1 kHz; that is, a sample is saved every 22.7 µs.
Hence, a pure sine wave of any frequency, or any recording, could be stored on a computer using any sampling rate, the quality of the recording determined by the sampling rate (the higher the better). [Technical note: Of course there is a lower limit (in some sense) on the sampling rate. This is called the Nyquist rate.]
To determine the mean frequency of the sound at any small time, you could use some advanced techniques from Fourier analysis, but it is not entirely trivial.